Gelatin-transglutaminase hemostatic dressings and sealants

ABSTRACT

An adhesive material for medical use comprising gelatin and a non-toxic cross-linking material such as transglutaminase. An optional embodiment of the invention includes dressings in which a layer of a transglutaminase is sandwiched between a first and second layer of gelatin. The hemostatic products are useful for the treatment of wounded tissue.

FIELD OF THE INVENTION

The present invention relates to hemostatic dressings, devices, andagents that contain resorbable or non-resorbable materials and/orcoagulation proteins. The hemostatic devices are useful for thetreatment of wounded tissue.

BACKGROUND OF THE INVENTION

The control of hemorrhage (bleeding) is a critical step in first aid andfield trauma care. Unfortunately, the occurrence of excessive bleedingor fatal hemorrhage from an accessible site is not uncommon (J. M. Rockoet al. (1982). J. Trauma 22:635). Mortality data from the Vietnam Warindicates that 10% of combat deaths were due to uncontrolled extremityhemorrhage. Up to one third of the deaths from exsanguination during theVietnam War could have been prevented by the use of effective fieldhemorrhage control methods. (SAS/STAT Users Guide, 4th ed. (Cary, N. C.:SAS Institute Inc; 1990)).

Although civilian trauma mortality statistics do not provide exactnumbers for pre-hospital deaths from extremity hemorrhage, case andanecdotal reports indicate similar occurrences (J. M. Rocko et al.(1982). J. Trauma 22:635). These data suggest that a substantialincrease in survival can be affected by the prehospital use of a simpleand effective method of hemorrhage control. Unfortunately, such a methodhas not been successfully demonstrated by use of commercially availablehemostatic devices.

Separately, surgical wound closure is currently achieved by sutures andstaples that facilitate healing by pulling tissues together. However,very often they fail to produce the adequate seal necessary to preventfluid leakage. Thus, there is a large, unmet medical need for devicesand methods to prevent leakage following surgery, including leaks thatfrequently occur along staple and suture lines. Such devices and methodsare needed as an adjunct to sutures or staples to achieve hemostasis orother fluid-stasis in peripheral vascular reconstructions, durareconstructions, thoracic, cardiovascular, lung, neurological, andgastrointestinal surgeries.

Most high-pressure hemostatic devices currently on the market arenominally, if at all adhesive. Good examples of such devices are theQuikClot® ACS™ (Z-Medica, Wallington, Conn.) and HemCon™ bandage(HemCon, Portland, Oreg.), the two hemostatic devices currently suppliedto members of the US armed forces. The mineral zeolite crystals in theQuikClot sponge cause adsorption of the water molecules in the blood,thus concentrating the clotting factors and accelerating blood clotting.The chitosan mixture that makes up the HemCon bandage has a positivecharge and attracts red blood cells, which have a negative charge. Thered blood cells are drawn into the dressing, forming a seal over thewound, and stabilizing the wound surface.

The HemCon bandage product mentioned above was developed in an attemptto provide pre-hospital hemorrhage control and has already demonstratedlimited success in the field. However, the chitosan network that makesup the HemCon bandage can be saturated with blood and fail quickly whenfaced with brisk flood flow or after 1-2 hours when confronted withmoderate blood flow from a wound (B. S Kheirabadi et al. (2005). J.Trauma. 59:25-35; A. E. Pusateri et al. (2006). J. Trauma. 60:674-682).Also, the HemCon bandage patch is available only as a stiff patch thatcannot fit easily into irregular wounds, further limiting its utility.

Other polysaccharide-based hemostatic devices that have been suggestedfor use in hemorrhage control are RDH™ (Acetyl Glucosamine), TraumaDEX™(MPH), and Chitoskin™ (Chitosan & Gelatin). However, none of these typesof bandages have been able to consistently demonstrate avoidance offailure in the face of significant blood flow. Other recently introducedhemostatic devices include Celox™ (Chitosan Crystals) and WoundStat™(TraumaCure Inc., MD) (granular blend of smectite mineral and a superabsorbent polymer). However, both of these products rapidly swell tofill wound sites, making them appropriate only for accelerating bloodclotting in specific types of wounds and presenting a danger of reducingor even eliminating blood flow in surrounding blood vessels.

QuikClot ACS™, also mentioned above, has also demonstrated efficacy instaunching moderate levels of hemorrhage. However, the water adsorptionmechanism of mineral zeolite cannot occur without the release of a largeamount of heat. As such, application of the QuikClot ACS™ results inhigh temperatures and severe burns at the injury site, which damagesurrounding tissue areas and make later medical care far morecomplicated (A. E. Pusateri et al. (2006). J. Trauma. 60:674-682).Clearly, a hemostatic solution without this significant side effect ismore ideal. While QuikClot has developed a mineral mixture that releasesless heat upon application, the efficacy of the cooler mixture isinsufficient for serious trauma care. Furthermore, neither the originalnor cooler mineral mixtures can stop brisk arterial bleeding.

All of the above-mentioned products rely on the natural clotting cascadeto control fluid leakage from a wound site. As such, they are all onlyuseful only for stopping blood flow and each only under conditionsappropriate for that particular device. General wound site sealing,particularly of injured sites leaking non-blood fluids, are beyond thescope of these products.

SUMMARY OF THE INVENTION

There is a need for, and it would be useful to have, a non-toxicadhesive material which could be used for a wide variety ofapplications, including but not limited to surgical applications,control of hemorrhage and control of bleeding from a wound. There isalso a need for, and it would be useful to have, a non-toxic adhesivematerial which could be used as part of a hemostatic bandage. There isalso a need for, and it would be useful to have, a non-toxic adhesivematerial which could be used as a surgical sealant.

The present invention overcomes the drawbacks of the background art byproviding an adhesive material which comprises a cross-linkable proteinand a non-toxic material which induces cross-linking of thecross-linkable protein. Preferably, the cross-linkable protein includesgelatin and any gelatin variant or variant protein as described herein.Optionally and preferably, the non-toxic material comprisestransglutaminase (TG), which may optionally comprise a microbialtransglutaminase (mTG). According to some embodiments of the presentinvention, the adhesive material is provided in a bandage, which ispreferably adapted for use as a hemostatic bandage. According to otherembodiments, it is provided as a sealant, which is preferably adaptedfor use as a surgical sealant.

When acted upon by a transglutaminase, gelatin, which is a denaturedform of the protein collagen, undergoes rapid crosslinking to form avibrant gel. The gelation process that takes place is extremely similarto the natural late stage clotting cascade that fibrin undergoes when itcomes into contact with Factor XIII and calcium. Furthermore, theresulting gel demonstrates adhesive capacity very similar to, if notgreater than, that of fibrin glues. (M. K. McDermott, Biomacromolecules.2004 July-August; 5(4):1270-9).

The present invention utilizes the similarities of gelatin-TGcross-linking to the fibrin clotting cascade to mimic the superiorhemostatic performance of an advanced fibrin dressing. Replacing thefibrin-thrombin sandwich of a fibrin bandage with a gelatin-TG sandwichresults in the creation of a novel, inexpensive, and stable dressingthat can control hemorrhage without significant side effects. This newbandage maximizes the adhesive properties of the gelatin-TG mixture byallowing for the controlled application of a large amount of the mixtureto a wound site in a way that prevents the spread of TG to areas notcontacted by the bandage. As such, this synergistic technologyrepresents an advance to both the field of advanced fibrin dressings andthe field of gelatin-TG adhesion.

Unlike a clotted fibrin network, the gelatin-TG network has anadditional benefit in that it can be dissolved specifically using aspecified protease that is not otherwise physiologically reactive (T.Chen, Biomacromolecules. 2003 November-December; 4(6):1558-63). Thus,while a gelatin-mTG hemostatic sandwich dressing can replicate theperformance of a fibrin-thrombin hemostatic sandwich dressing, it canalso be removed as desired without complication.

Beyond its application as a hemostatic field-dressing for trauma care,the present invention of a gelatin-TG based hemostatic device has greatpotential in controlling brisk, arterial bleeding during surgery,bleeding after endo-vascular catherization, or leakage of other bodilyfluids after injuries or doing surgery.

To date, though the gelling properties of cross-linked gelatin-TG andthe adhesive capacity of gelatin-TG cross-linking have been separatelyexplored, no efforts have been made to use both characteristics togetherto form a hemostatic or tissue sealing composition.

Adhesive use of the gelatin-TG compound was demonstrated in vivo in arat retina model, where a drop of gelatin-TG mixture was used forretinal attachment (T. Chen, J Biomed Mater Res B Appl Biomater. 2006May; 77(2):416-22).

Use of gelatin-TG gel as a scaffold for cell therapy was also tested(U.S. Pat. No. 5,834,232. Also Ito A, J Biosci & Bioeng. 2003; 95(2):196-99. Also, Broderick E P, J Biomed Mater Res B Appl Biomater. 2005Jan. 15; 72(1):37-42).

While these studies emphasized the safety of physiological use of thegelatin-TG mixture, they each only used one of the characteristics ofgelatin-TG cross-linking, and failed to teach or suggest the hemostasisand tissue sealing advancement presented in the current invention.

Use of the gelatin-TG mixture for hemostasis or fluid-stasis also marksa significant advancement from a number of well-documented attempts touse transglutaminases, particularly tissue TG, independently as surgicaladhesives (U.S. Pat. No. 5,736,132, U.S. 61/908,196, among many).Employing gelatin as a substrate for TG adds a mechanical scaffold tothe TG activity that provides a number of advantages over the use of TGalone. TG together with gelatin can be applied more precisely than TGalone, can conform precisely to a wound site, and allows for controlledbioabsorbability.

The present invention overcomes the drawbacks of the background art.Prior attempted solutions used many forms of modified and unmodifiedgelatin networks for mild to moderate hemostasis. However, a method offorming, in situ, a strongly cross-linked gelatin network that cancontrol brisk bleeding arterial hemorrhage or other significant bodilyfluid leakages has been lacking. A method, such as gelatin-TGcross-linking that can form a strong gelatin network in vivo increasesthe mechanical strength of a gelatin matrix and makes it suitable forcontrolling high-pressure arterial bleeding and other bodily fluidleakages. Aside from the improved method of cross-linking, the presentinvention involves many other innovations that provide it withadvantages over existing gelatin-based hemostatic materials. Anon-limiting, illustrative, partial list is provided below:

-   -   1) In-situ cross-linking between gelatin chains and endogenous        collagen of tissue ECM (extra cellular matrix) creates a strong,        hemostatic barrier for fluids.    -   2) Gelatin and TG can more effectively affect hemostasis or        fluid-stasis by being applied in lyophilized form and        reconstituted by the blood or other body fluid.    -   3) A gelatin-TG mixture in lyophilized form has increased shelf        life.    -   4) Gelatin and TG in layered, lyophilized form provided more        rapid reconstitution, which is helpful for a high pressure fluid        flow environment.    -   5) The addition of a mechanical backing to the basic gelatin-TG        mixture increases the hemostatic or fluid control capacity of        the mixture by slowing the fluid and allowing the gelatin-TG        more time to cross-link and block the fluid leakage.

According to some preferred embodiments of the present invention, thegelatin-mTG mixture is partially cross-linked prior to application to awound site or prior to lyophilization. In another embodiment,non-cross-linked gelatin or mTG is present together with partiallycross-linked gelatin-mTG.

Hemostatic bandages which are adhesive in nature are known in the art,yet have many complications and drawbacks to their use. For example, thewidespread hemostatic use of fibrinogen and thrombin was common in thelast year of World War II, but was abandoned because of the transmissionof hepatitis (D. B. Kendrick, Blood Program in WW II (Washington, D.C.:Office of the Surgeon General, Department of Army; 1989), 363-368).

Fibrinogen dressings were first used by trauma surgeons during World WarI when Grey and his colleagues made prepolymerized fibrin sheets andpowders. During World War II, fibrin glue was created withprepolymerized Styrofoam-like sheets of fibrin and fibrin films by theUnited States military and the American Red Cross. Fibrin baseddressings show a significant difference in controlling bleeding time andreducing blood loss when compared to a control. (Jackson, M., et al.(1996). J. of Surg. Res. 60:15-22; and Jackson, M., et al. (1997). Surg.Forum. XL, VIII:770-772)

Despite the efficacy of fibrinogen dressings in controlling hemorrhage,the use of fibrinogen dressings was discontinued as blood and serumborne diseases such as hepatitis and HIV were often transmitted sincethe dressings comprised purified human or animal fibrinogen or otherpurified blood products. (Holcomb, J. B., et al. (1997). SurgicalClinics of North America. 77:943-952)

In the past few years, however, there has been a renewed interest infibrin based products for treating wounds as plasma purificationtechniques have greatly reduced the risk of blood and serum bornediseases.

A hemostatic sandwich dressing has been described by the US Red Cross,which contains a layer of thrombin sandwiched between layers offibrinogen (see, e.g., PCT/US99/10952, U.S. Pat. Nos. 6,054,122,6,762,336). That hemostatic dressing has demonstrated much success intreating potentially fatal trauma wounds (E. M. Acheson. (2005). J.Trauma. 59(4):865-74; discussion 874-5; B. S. Kheirabadi. (2005). J.Trauma. 59(1):25-34; discussion 34-5; A. E. Pusateri. (2004). J. Biomed.Mater. Res. B Appl. Biomater. 15; 70(1):114-21) In fact, in thoseporcine studies, the fibrin sandwich dressing greatly outperformed theHemCon and QuikClot products in treating potentially fatal traumawounds, demonstrating a >75% survival rate after 2 hours, versus 0%survival when the standard army field bandage, HemCon bandage, orQuikClot powder was used.

Although such dressings can be used in methods for treating woundedtissue, such conventional sandwich dressings can become delaminated,whereby the edges of the layers of the dressing no longer adhere to eachother. Such delamination can result in reduced interaction of thedressing components layers, with decreased effectiveness of the dressingin preventing hemorrhage.

An improved fibrin-based hemostatic sandwich dressing has been describedwhich comprises a plurality of layers that contain resorbable materialsand/or coagulation proteins. Specifically, the dressing (seePCT/US03/28100, U.S. Patent Application No. 60/155,234) includes a layerof thrombin sandwiched between a first and second layer of fibrinogen,wherein the layer of thrombin is not coextensive with the first and/orsecond layer of fibrinogen.

Despite the advances in fibrin wounds dressings, these bandages sufferfrom many drawbacks. The lyophilized fibrinogen used to make the bandagemust be purified from human blood plasma. As this is a costly anddelicate procedure, the resulting fibrinogen bandage is extremelyexpensive to produce and only has a very short shelf life at roomtemperature. The more fibrinogen that is added to the backing, thebetter the bandage works in stopping bleeding. However, the morefibrinogen added to the backing, the more costly the bandage.Additionally, high amounts of fibrinogen on the bandage backing maycontribute to the fragility of the bandage, making it crumbly anddifficult to work with. As a result of these limitations, no efficaciousfibrin bandage is commercially available.

Thus, while an advanced fibrin dressing could control hemorrhage withoutsignificant side effects and fill the previously mentioned deficiency inactive trauma care hemostasis, price and stability limitations presentstrong disadvantages to the use of this type of dressing.

Liquid fibrin sealants or glues have been used for many years as anoperating room adjunct to hemorrhage control (J. L. Garza et al. (1990).J. Trauma. 30:512-513; H. B. Kram et al. (1990). J. Trauma. 30:97-101;M. G. Ochsner et al. (1990). J. Trauma. 30:884-887; T. L. Matthew et al.(1990). Ann Thorac. Surg. 50:40-44; H. Jakob et al. (1984). J. Vasc.Surg. 1:171-180). Also, single donor fibrin sealants have also beenwidely used clinically in various surgical situations. (W. D. Spotnitz.(1995). Thromb. Haemost. 74:482-485; R. Lerner et al. (1990). J. Surg.Res. 48:165-181)

While a number of absorbable surgical hemostats are currently used inthe surgical arena, no existing product is sufficiently strong toprovide the mechanical and biological support necessary to controlsevere hemorrhage or vigorous flow of other biological fluids.

Currently available hemostatic bandages such as collagen wound dressings(INSTAT™, Ethicon, Somerville, N.J., and AVITENE™, C R Bard, MurrayHill, N.J.) or dry fibrin thrombin wound dressings (TACHOCOMB™, HafslundNycomed Pharma, Linz, Austria) are restricted to use in surgicalapplications, and are not sufficiently resistant to dissolution in highblood flow. They also do not possess enough adhesive properties to serveany practical purpose in the stanching of severe blood flow. Thesecurrently available surgical hemostatic bandages are also delicate andthus prone to failure should they be damaged by bending or loading withpressure. They are also susceptible to dissolution in hemorrhagicbleeding. Such dissolution and collapse of these bandages may becatastrophic, because it can produce a loss of adhesion to the wound andallow bleeding to continue unabated.

Arterial bleeding is also not manageable with the application ofoxidized cellulose (SURGICEL, Ethicon, Somerville, N.J.) or gelatinsponge (SURGIFOAM, Ethicon, Somerville, N.J.) absorbable hemostats.These products are intended to control low-pressure bleeding from boneand epidural venous oozing. Gelatin sponges are not appropriate forhigh-pressure, brisk flowing arterial bleeding because they do not forma tight bond with the source of bleeding and are thus easily dislodged.Oxidized cellulose is also not appropriate for controlling arterialbleeding because it swells and needs to be removed from the applicationsite when hemostasis is achieved. When the blood flow is too high, toomuch swelling occurs before hemostasis can be achieved (M. Sabel et al.(2004). Eur. Spine J. 13 (1):597-101).

The most widely used tissue adhesives are generally unfit for use ashemostatic or internal fluid-stasis devices, for reasons generallyrelated to mild toxicity and inability to be easily prepared and appliedin the field. A good example of this is the cyanoacrylate family oftopical skin adhesives, such as Dermabond™, Indermil™, Liquiband™ etc.The nature of cyanoacrylate's rapid activation when exposed to airrenders cyanoacrylate-based products inappropriate for use in an activehemostatic field dressing and their inability to bind to wet surfacesrenders them inappropriate for internal hemostatis or fluid-stasisusage.

Existing products that are intended for internal fluid-stasis usage alsohave significant problems. BioGlue™ (Cryolife Inc.) is a strong adhesiveand sealant but contains albumin crosslinked by glutaraldehyde, asubstance which is toxic and highly neurotoxic. This toxicity greatlylimits its usage. Another sealant is CoSeal (Baxter), which is composedof polyethylene glycol (PEG). Though it is non-toxic, it has only weakadhesive strength, greatly limiting its applications.

Gelatin has been used in a variety of wound dressings. Since gelatingels have a relatively low melting point, they are not very stable atbody temperature. Therefore, it is imperative to stabilize these gels byestablishing cross-links between the protein chains. In practice, thisis usually obtained by treating the gelatin with glutaraldehyde orformaldehyde. Thus, cross-linked gelatin may be fabricated into drysponges which are useful for inducing hemostasis in bleeding wounds.Commercially available examples of such sponges include Spongostan(Ferrosan, Denmark), Gelfoam (Upjohn, USA), and Surgifoam (Ethicon.Somerville, N.J.). A major disadvantage of these sponges is that thecross-linking agent used (formaldehyde or glutaraldehyde) is toxic forcells. The negative effect of glutaraldehyde cross-linking isexemplified, for instance, by the findings of de Vries et al (AbstractBook of the Second Annual Meeting of the WHS, Richmond, USA, p 51,1992). These authors showed that glutaraldehyde cross-linked collagenlattices were toxic for cells, whereas the non cross-linked variety wasnot. Therefore, despite their beneficial hemostatic properties, theseproducts are not very optimal as wound dressings for the treatment ofproblematic wounds. Consequently, a gelatin-based wound dressing whichuses a different, less toxic, cross-linking technology would be verydesirable.

Aside from potential toxicity, gelatin networks alone do not provide themechanical properties necessary for controlling brisk bleeding. They aremore appropriate for wound management applications that only require asmall amount of fluid absorption. In one study, it was concluded thatsheets of glutaraldehyde cross-linked gelatin are more appropriate as adressing for sustained wound healing, particularly of dystrophic tissuewhich need longer time. Alternatively, they may be useful as a scaffoldfor cell attachment, where they can stimulate a poorly reactivemicroenvironment throughout prolonged in situ presence (M G Tucci.(2001). J. Bioactive & Comp. Polymers. 16(2): 145-157).

Gelatin networks cross-linked with polysaccharides have also beensuggested for use in controlling bleeding. These hemostatic compoundsare unhindered by the potential toxicity of glutaraldehyde cross-linkedgelatin sponges. However, the gelatin-polysaccharide substancesgenerally lack mechanical strength and are intended mainly to controlsmall amounts of oozing fluid during surgery or to limit wound oozingover an extended, post-medical care period.

One example of a gelatin-polysaccharide compound is a gelatin-alginatewound dressing that is cross-linked in situ. Such a dressing has noadhesive function and is mainly used to hold in moisture on the woundsite. The dressing swells to 90% of its initial size, which greatlyreduces its mechanical strength (B Balakrishnan et al. (2005).Biomaterials. 26(32):6335-42).

Another, more widespread example, is a cross-linked gelatin-chitosanwound dressing (examples in U.S. Pat. Nos. 6,509,039, 4,572,906). Whilesome have suggested the use of such dressings for trauma care(Chitoskin™), the hemostatic properties of this material are simplyinsufficient to control high-pressure bleeding. Also, the materialswells significantly when confronted with high volumes of bodily fluids.Such dressings are more appropriate for treating chronic wounds andburns.

Yet another example is mentioned (U.S. Pat. No. 6,132,759) wheresolubilized gelatin is cross-linked with oxidized dextran. This materialis suggested for the covering and long-term treatment of wounds since itdemonstrated a high absorptive capacity and favorable controlled releaseproperties for the delivery of therapeutic substances, particularly towounds.

Currently no material involving cross-linked gelatin networks ornetworks of other materials cross-linked with gelatin has been able toindependently provide hemostasis for brisk internal bleeding, even withthe addition of thrombin. A study was done comparing the hemostaticcapacity of FloSeal gelatin matrix (BioSurgery, Fremont, Calif.) andGelFoam gelatin matrix soaked in active thrombin solution. Neitherenhanced hemostatic device was able to stop flow characterized bleed inmore than ⅔ of patients after 5 minutes. Pulsatile arterial bleeding isfar more brisk than flow bleeding and would most certainly present aproblem for these thrombin-soaked matrices (F A Weaver et al. (2002).Ann Vasc. Surg. 16(3):286-93).

In any case, there remains a distinct deficiency in trauma care, in thatthere is no novel, active hemostatic field dressing available that cancontrol hemorrhage without significant side effects. Similarly, thereremains a distinct deficiency in surgical care, in that there is nonon-toxic sealant available that is capable of withstanding briskbleeding and able to seal wound sites leaking non-blood body fluids.

According to some embodiments of the present invention, there isprovided a method of treating a wounded tissue, comprising applying tothe tissue a composition comprising gelatin and a non-toxiccross-linking agent.

Optionally, the non-toxic cross-linking agent comprisestransglutaminase. Preferably, the transglutaminase is included as partof a transglutaminase composition and the weight ratio of gelatin totransglutaminase composition is in a range of from about 1:1 to about300:1. More preferably, the transglutaminase composition has a specificactivity level of at least about 40 U/gm. Most preferably, thetransglutaminase composition has a specific activity level of at leastabout 800 U/gm.

Optionally and preferably, activity of the transglutaminase in thegelatin-transglutaminase composition is from about 25 to about 400 U/gof gelatin. More preferably, the activity is from about 40 to about 200U/g of gelatin.

Optionally, the transglutaminase comprises a plant, recombinant animal,or microbe derived transglutaminase other than blood derived FactorXIII. Preferably, the composition has a pH in a range of from about 5 toabout 8.

Optionally, the gelatin is produced from animal origin, recombinantorigin or a combination thereof. Preferably, the animal origin isselected from the group consisting of fish and mammals. More preferably,the mammal is selected from the group consisting of pigs and cows.

Optionally, the gelatin is of type A (Acid Treated) or of type B(Alkaline Treated). More preferably, gelatin comprises high molecularweight gelatin.

Optionally, wounded tissue is selected from the group consisting ofsurgically cut tissue, surgically repaired tissue, and traumatizedtissue.

Optionally, the method further comprises reducing bleeding or leakage ofother bodily fluids from the tissue. Optionally a bodily fluid isselected from the group consisting of cerebral spinal fluid, intestinalfluid, air, bile, and urine. Preferably, the method further comprisesinducing hemostasis or stasis of other leaking bodily fluids in thetissue.

Optionally, the wound is bleeding or leaking another bodily fluid andtreating the wounded tissue comprises applying the composition to thewound site to encourage in situ cross-linking between gelatin chains andthe endogenous collagen of tissue extracellular matrix to create abarrier to fluid leakage or bleeding.

Optionally, the method further comprises forming a biomimetic clot.

Optionally, applying the composition comprises: Mixing the gelatin andthe transglutaminase to form a mixture; and Applying the mixture to thetissue.

According to other embodiments of the present invention, there isprovided a method for inducing hemostasis in a wound of a mammal, themethod comprising applying to the wound a composition comprising gelatinand transglutaminase.

According to still other embodiments of the present invention, there isprovided a method for inducing formation of a biomimetic clot at a siteof a damaged blood vessel, comprising applying to the wound acomposition comprising gelatin and transglutaminase.

According to still other embodiments of the present invention, there isprovided a composition comprising a combination of gelatin andtransglutaminase, wherein a ratio of an amount of the gelatin and anamount of the transglutaminase is selected to induce formation of abiomimetic clot in a mammal.

According to still other embodiments of the present invention, there isprovided a composition comprising a combination of gelatin and non-toxiccross-linking agent, wherein a ratio of an amount of the gelatin and anamount of the non-toxic cross-linking agent is sufficient to reducebleeding in a wound of a mammal.

Preferably, the non-toxic cross-linking agent comprisestransglutaminase. More preferably, the transglutaminase is added as partof a transglutaminase composition and the weight ratio of gelatin totransglutaminase composition is in a range of from about 1:1 to about300:1. More preferably, the ratio is in a range of from about 1:1 toabout 100:1. Most preferably, the transglutaminase composition has aspecific activity level of at least about 40 U/gm. Also most preferably,the transglutaminase composition has a specific activity level of atleast about 80 U/gm. Also most preferably, the transglutaminasecomposition has a specific activity level of at least about 200, 400 or800 U/gm.

Optionally activity of the transglutaminase in thegelatin-transglutaminase composition is from about 25 to about 400 U/gof gelatin. Preferably, activity is from about 40 to about 200 U/g ofgelatin.

Optionally, the transglutaminase comprises a plant, recombinant, animal,or microbe derived transglutaminase other than blood derived FactorXIII. Preferably, the composition further comprises a stabilizer orfiller. Also preferably, the composition has a pH in a range of fromabout 5 to about 8.

Optionally, gelatin is produced from animal origin, recombinant originor a combination thereof. Preferably, the animal origin is selected fromthe group consisting of fish and mammals. More preferably, the mammal isselected from the group consisting of pigs and cows. Most preferably,the gelatin comprises pig skins or pig bones, or a combination thereof.Also most preferably, the gelatin is of type A (Acid Treated) or of typeB (Alkaline Treated). Also most preferably, the gelatin comprises highmolecular weight gelatin.

Optionally, the gelatin has a bloom of at least about 250. Preferably,the fish comprises a cold water species of fish.

Optionally, recombinant gelatin is produced using bacterial, yeast,animal, insect, or plant systems or any type of cell culture.

Optionally, gelatin is purified to remove salts.

Optionally, gelatin has at least one adjusted, tailored or predeterminedcharacteristic. Optionally, the gelatin does not undergothermoreversible gelation.

According to still other embodiments of the present invention, there isprovided a hemostatic or body fluid sealing agent comprising acombination of gelatin and a non-toxic cross-linking agent. Optionally,the non-toxic cross-linking agent comprises transglutaminase.Preferably, the combination comprises aggregated gelatin andtransglutaminase.

As described herein, a method or composition in which thetransglutaminase may optionally be extracted from one or more ofStreptoverticillium Baldaccii, a Streptomyces Hygroscopicus strain, orEscherichia Coli.

According to still other embodiments of the present invention, there isprovided a method of inducing hemostasis in and/or sealing a woundedtissue, comprising applying to the tissue a composition comprising across-linking protein substrate and a non-toxic cross-linking agent.Optionally, the non-toxic cross-linking agent comprisestransglutaminase. Preferably, the substrate comprises one or moresynthesized polymer sequences featuring a transglutaminase cross-linkingsite. More preferably, the substrate comprises a modified polypeptidecomprising at least one transglutaminase cross-linking site.

According to still other embodiments of the present invention, there isprovided a composition for inducing hemostasis and/or sealing a wound,comprising a mixture of gelatin and transglutaminase, wherein themixture is modified such that the gelatin forms a solution withtransglutaminase at a temperature lower than the natural sol-geltransition temperature of standard animal gelatin.

Optionally, the gelatin has been modified to have a reduced sol-geltransition temperature. Preferably the composition further comprises anadditive to increase solubility of the gelatin in the mixture. Morepreferably the composition further comprises an additive to reduce thesol-gel transition temperature of the gelatin. Most preferably, thecomposition further comprises a plasticizer. Optionally and mostpreferably, the plasticizer is selected from the group consisting of apolyhydric alcohol, glycerine, glycerol, xylitol, sucrose, sorbitol,triethanolamine, resorcin, thiodiglycol, sodium salt oftoluenesulphoacid, butylene glycol, urea nitrate, thiourea, urea,glutamic acid, aspargic acid, valine, glycine, KSCN, KI, and LiBr.

Optionally, a concentration ratio range for glycerol is from about 0.5:1to about 5:1 Glycerol:Gelatin, weight per weight. Preferably, theconcentration ratio range is from about 1:1 to about 2:1Glycerol:Gelatin, weight per weight. Optionally, a concentration ratiorange for sorbitol is from about 0.5:1 to about 5:1 Sorbitol:Gelatinweight per weight. Preferably, the concentration ratio range is fromabout 1:1 to about 3:1 Sorbitol:Gelatin, weight per weight.

Optionally, a concentration ratio range for urea is from about 1:2 toabout 2:2 urea:gelatin, weight per weight.

Optionally the composition further comprises an adjusting agent selectedfrom the group consisting of a pH adjusting agent and an ionconcentration adjusting agent. Preferably, the pH adjusting agentprovides a pH in a range of from about 1.5 to about 5.0 or from about7.0 to about 9.0.

Optionally the composition further comprises a salt.

Optionally, the composition further comprises a trehalose carbohydrate,mannitol carbohydrate, or other carbohydrate for stabilization for spraydrying, lyophilization, or other protein drying.

Optionally the composition further comprises a denaturant. Preferably,the denaturant is selected from the group consisting of GuanidineHydrochloride and Urea. More preferably a concentration ratio range isfrom about 1:2 to about 2:2 GuHCl:gelatin, weight per weight. Also morepreferably, a concentration ratio range is from about 0.5:1 to about 1:1urea: gelatin, weight per weight.

Optionally the composition further comprises a reducing agent.Preferably, the reducing agent is selected from the group consisting ofmagnesium chloride and hydroquinone. More preferably, the hydroquinoneis present in solution of the mixture at a concentration of from about0.2 to about 0.5 M. Most preferably, the concentration is from about 0.3to about 0.4 M.

Optionally, the magnesium chloride is present in solution of the mixtureat a concentration of from about 2 to about 4 M. Preferably theconcentration is from about 2.5 to about 3.5M.

Optionally the composition further comprises an exothermic agent.Preferably, the exothermic agent comprises one or more of calciumchloride, other calcium salts, magnesium chloride, metallicoxides/zeolites, or a combination thereof. More preferably the calciumchloride is present in an amount of from about 0.2 to about 0.7 g ofCalcium Chloride per mL of the mixture in solution for each degreeCelsius increase in temperature above the ambient temperature.

Optionally the composition further comprises a gelatin specificprotease.

Optionally the composition further comprises a protease inhibitor.

Optionally the composition further comprises an additional hemostaticagent. Preferably the additional hemostatic agent further comprises oneor more of albumin, collagen, fibrin, thrombin, chitosan, ferricsulfate, or other metal sulfates.

According to other embodiments of the present invention there isprovided a hemostatic or sealing dressing which comprises: (i) a firstgelatin layer; (ii) a transglutaminase layer adjacent to the firstgelatin layer; and (iii) a second gelatin layer adjacent to thetransglutaminase layer, wherein the transglutaminase layer iscoextensive or noncoextensive with the first gelatin layer and/or thesecond gelatin layer.

According to other embodiments of the present invention there isprovided a hemostatic or sealing dressing which comprises: (i) aresorbable or non-resorbable material layer; (ii) a first gelatin layeradjacent to the material layer; (iii) a transglutaminase layer adjacentto the first gelatin layer; and (iv) a second gelatin layer adjacent tothe transglutaminase layer, wherein the transglutaminase layer iscoextensive or noncoextensive with the first gelatin layer and/or thesecond gelatin layer.

According to other embodiments of the present invention there isprovided a hemostatic or sealing dressing which comprises: (i) a gelatinlayer; (ii) a transglutaminase layer adjacent to the gelatin layer;wherein the transglutaminase layer is coextensive or noncoextensive withthe gelatin layer.

According to other embodiments of the present invention there isprovided a hemostatic or sealing dressing which comprises: (i) aresorbable or non-resorbable material layer; (ii) a gelatin layeradjacent to the material layer; (iii) a transglutaminase layer adjacentto the gelatin layer; wherein the transglutaminase layer is coextensiveor noncoextensive with the gelatin layer.

According to other embodiments of the present invention there isprovided a hemostatic or sealing dressing which comprises: (i) a gelatinlayer; (ii) a resorbable or non-resorbable material layer adjacent tothe first gelatin layer; (iii) a transglutaminase layer adjacent to thematerial layer; wherein the transglutaminase layer is coextensive ornoncoextensive with the gelatin layer.

Optionally, the dressing further comprises a backing material.

According to other embodiments of the present invention there isprovided a hemostatic or sealing device which comprises: (i) aresorbable or non-resorbable matrix; (ii) gelatin; (iii) atransglutaminase; wherein the gelatin and transglutaminase areincorporated within the matrix.

According to other embodiments of the present invention there isprovided a hemostatic or sealing device which comprises: (i) a porousresorbable or non-resorbable matrix; (ii) gelatin; (iii) atransglutaminase; wherein the gelatin and transglutaminase are adheredto the matrix.

According to other embodiments of the present invention there isprovided a medical device for insertion into a body of a human or lowermammal, comprising a hemostatic or sealing agent or composition asdescribed herein. Preferably the device comprises a vascular catheter.

According to other embodiments of the present invention there isprovided a medical device for topical application on the body of a humanor lower mammal, comprising a hemostatic or sealing agent or compositionas described herein. Optionally the device comprises a pressurized sprayor foam.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are intended to provide further explanation of the invention asclaimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. All patents, patent applications,and publications mentioned herein are incorporated herein by reference.

As used herein, a transglutaminase layer that is said to be“noncoextensive” with a gelatin layer is one in which the spatialboundaries of the transglutaminase layer in two dimensions are smallerthan the spatial boundaries of one or both gelatin layers such that thetransglutaminase layer is coextensive with only about 5% to about 95% ofthe surface area of the first gelatin layer of the hemostatic dressingand/or coextensive with only about 5% to about 95% of the surface layerof the second gelatin layer of the hemostatic dressing, independently.For example, the transglutaminase layer can be coextensive with about10, 20, 30, 40, 50, 60, 70, 75, 80, or 90% of the surface area of eachof the first and second gelatin layers, independently. Atransglutaminase layer that is “coextensive” with a gelatin layerprovides full coverage of the gelatin layer and is coextensive with 100%of the surface area of the gelatin layer. A transglutaminase layer canbe noncoextensive with the first gelatin layer and yet be coextensivewith the second gelatin layer, or vice versa, e.g., by employing gelatinlayers having different total surface areas or shapes.

“Patient” as used herein refers to human or animal individuals in needof medical care and/or treatment.

“Wound” as used herein refers to any damage to any tissue of a patientthat results in the loss of blood from the circulatory system or theloss of any other bodily fluid from its physiological pathway. Thetissue can be an internal tissue, such as an organ or blood vessel, oran external tissue, such as the skin. The loss of blood or bodily fluidcan be internal, such as from a ruptured organ, or external, such asfrom a laceration. A wound can be in a soft tissue, such as an organ, orin hard tissue, such as bone. The damage may have been caused by anyagent or source, including traumatic injury, infection or surgicalintervention. The damage can be life-threatening ornon-life-threatening.

“Resorbable material” as used herein refers to a material that is brokendown spontaneously and/or by the mammalian body into components whichare consumed or eliminated in such a manner as not to interferesignificantly with wound healing and/or tissue regeneration, and withoutcausing any significant metabolic disturbance.

“Stability” as used herein refers to the retention of thosecharacteristics of a material that determine activity and/or function.

“Binding agent” as used herein refers to a compound or mixture ofcompounds that improves the adherence of one layer of the hemostaticdressing to one or more different layers and/or the adherence of thecomponents of a given layer to other components of that layer.

“Solubilizing agent” as used herein refers to a compound or mixture ofcompounds that improves the dissolution of a protein or proteins in a(preferably) aqueous solvent.

“Filler” as used herein refers to a compound or mixture of compoundsthat provide bulk and/or porosity to one or more layers of thehemostatic dressings.

“Release agent” as used herein refers to a compound or mixture ofcompounds that facilitates removal of an hemostatic dressing from amanufacturing mold.

“Foaming agent” as used herein refers to a compound or mixture ofcompounds that produces gas when hydrated under suitable conditions.

“TG” refers to transglutaminase of any type; “mTG” may also refer tomicrobial transglutaminase and/or to any type of transglutaminase,depending upon the context (in the specific experimental Examples below,the term refers to microbial transglutaminase).

The term “mammal”, particularly with regard to method of treatmentand/or use or application of a device and/or composition, refers to bothhumans and lower mammals, unless otherwise specified.

As used herein, “about” means plus or minus approximately ten percent ofthe indicated value.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin order to provide what is believed to be the most useful and readilyunderstood description of the principles and conceptual aspects of theinvention. In this regard, no attempt is made to show structural detailsof the invention in more detail than is necessary for a fundamentalunderstanding of the invention, the description taken with the drawingsmaking apparent to those skilled in the art how the several forms of theinvention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic block diagram of an exemplary bandage according tothe present invention;

FIG. 2 shows a frontal view of an exemplary bandage according to thepresent invention, covered with an optional absorbable backing and anoptional plastic wrapping;

FIG. 3 is a schematic block diagram of an exemplary of a hemostaticdevice according to the present invention, incorporating a porousmatrix;

FIG. 4 is a graph showing the effect of different percentages of atested gelatin on wound strength;

FIG. 5 shows effect of temperature on activity of transglutaminase(Temperature Range tested was 32 to ˜150° F.; optimum range was 122-131°F. (50-55° C.));

FIG. 6 shows representative burst pressure measurements of tissueadhesives based on composition A;

FIG. 7 shows representative burst pressure measurements of tissueadhesives based on composition B;

FIG. 8 is a photograph showing the formation of the gel and alsoinduction of hemostasis (FIG. 8A shows the entire area while FIG. 8Bshows a portion of the area, magnified for further details);

FIG. 9A shows lack of clot formation after application of controlsolution, while FIG. 9B shows gelation of the experimental solution andhemostasis;

FIGS. 10, 10C and 10D (note that the Figure labeled “FIG. 10” includes“A” and “B” labels in the photographs themselves) show photographs ofthe artery as it was being cut (see photograph labeled “A”); the cutartery, bleeding profusely (see photograph labeled “B”); application ofthe composition of the present invention to the cut artery (10C); andhemostasis, with formation of a biomimetic clot (10D);

FIGS. 11A and 11B show an example of the double syringe method ofapplication of the two component hemostatic sealant; and

FIGS. 12A and 12B show an example of a vascular insertion point closurewhere the catheter is covered by the components described herein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is of an adhesive material which comprises across-linkable protein and a non-toxic material which inducescross-linking of the cross-linkable protein. Preferably, thecross-linkable protein includes gelatin and any gelatin variant orvariant protein as described herein. Optionally and preferably, thenon-toxic material comprises transglutaminase (TG), which may optionallycomprise any type of calcium dependent or independent transglutaminase(mTG), which may for example optionally be a microbial transglutaminase.According to some embodiments of the present invention, the adhesivematerial is provided in a bandage, which is preferably adapted for useas a hemostatic bandage. Various embodiments of the present inventionare described in greater detail below, under section headings which areprovided for the sake of clarity only and without any intention of beinglimiting in any way.

Gelatin and Transglutaminase

According to preferred embodiments of the present invention, there isprovided a composition for hemostasis and tissue sealing in which thecross-linking material comprises transglutaminase and the cross-linkableprotein comprises gelatin.

According to a preferred embodiment, transglutaminase is present in acomposition having a specific activity level of at least about 100 U/gm,although optionally lower activity levels may also be used, for exampleby optionally adjusting the above described ratios. Such optionallylower activity levels of the composition preferably comprise at leastabout 20 U/gm, more preferably at least about 40 U/gm, even morepreferably at least about 60 U/gm and most preferably at least about 80U/gm.

The transglutaminase, whether alone or as part of a composition, ispreferably added to gelatin in an amount such that the resultingtransglutaminase activity in the mixture is preferably from about 25 toabout 100 U/g of gelatin and more preferably from about 40 to about 60U/g of gelatin.

Suitable gelatin and transglutaminase can be obtained by any of themethods known and available to those skilled in the art. Gelatin mayoptionally comprise any type of gelatin which comprises protein that isknown in the art, preferably including but not limited to gelatinobtained by partial hydrolysis of animal tissue and/or collagen obtainedfrom animal tissue, including but not limited to animal skin, connectivetissue (including but not limited to ligaments, cartilage and the like),antlers or horns and the like, and/or bones, and/or fish scales and/orbones or other components; and/or a recombinant gelatin produced usingbacterial, yeast, animal, insect, or plant systems or any type of cellculture.

According to preferred embodiments of the present invention, gelatinfrom animal origins preferably comprises gelatin from mammalian originsand more preferably comprises one or more of pork skins, pork and cattlebones, or split cattle hides, or any other pig or bovine source. Morepreferably, such gelatin comprises porcine gelatin since it has a lowerrate of anaphylaxis. Gelatin from animal origins may optionally be oftype A (Acid Treated) or of type B (Alkaline Treated), though it ispreferably type A.

Preferably, gelatin from animal origins comprises gelatin obtainedduring the first extraction, which is generally performed at lowertemperatures (50-60° C., although this exact temperature range is notnecessarily a limitation). Gelatin produced in this manner will be inthe range of 250-300 bloom and has a high molecular weight of at leastabout 95-100 kDa. Preferably, 300 bloom gelatin is used.

A non-limiting example of a producer of such gelatins is PB Gelatins(Tessenderlo Group, Belgium).

According to some embodiments of the present invention, gelatin fromanimal origins optionally comprises gelatin from fish. Optionally anytype of fish may be used, preferably a cold water variety of fish suchas carp, cod, or pike, or tuna. The pH of this gelatin (measured in a10% solution) preferably ranges from 4-6.

Cold water fish gelatin forms a solution in water at 10° C. and thus allcold water fish gelatin are considered to be 0 bloom. For the currentinvention, a high molecular weight cold water fish gelatin is preferablyused, more preferably including a molecular weight of at least about95-100 kDa. This is equivalent to the molecular weight of a 250-300bloom animal gelatin. Cold water fish gelatin undergoes thermoreversiblegelation at much lower temperatures than animal gelatin as a result ofits lower levels of proline and hydroxyproline. Per 1000 amino acidresidues, cold water fish gelatin has 100-130 proline and 50-75hydroxyproline groups as compared to 135-145 proline and 90-100hydroxyproline in animal gelatins (Haug I J, Draget K I, Smidsrød O.(2004). Food Hydrocolloids. 18:203-213).

A non-limiting example of a producer of such a gelatin is NorlandProducts (Cranbury, N.J.).

In a preferred embodiment of the invention, the gelatin is purified toremove salts. This can be accomplished according to previously describedtechniques. One such technique involves forming a 20% w/v solution ofgelatin in water and heating it to 60° C. under stirring. The mixture isthen let to stand still overnight. The gel obtained is dialysed againstrepeated changes of deionized water to eliminate salts, stirred andheated to 50° C. to disaggregate the physical network. The finalsolution was filtered and freeze-dried. (Crescenzi V, Francescangeli A,Taglienti A. (2002). Biomacromolecules. 3:1384-1391). Alternatively, thegelatin can be desalted by size exclusion column.

According to some embodiments of the present invention, a recombinantgelatin is used. Recombinant gelatins are currently commerciallyproduced by FibroGen (San Francisco, Calif.). The currently preferredmethod is using a recombinant yeast system (Pichia Pastoris) to expressspecified fragments of Type I, alpha1 human sequence collagen.

In an optional but preferred embodiment of the present invention,recombinant gelatins are fully synthetic molecules, containing nocontaminating components from humans or any animals. By “synthetic” itis meant that the gelatin is preferably produced according to a methodselected from chemical synthesis, cell free protein synthesis, celltissue culture, any type of bacterial, insect or yeast culture, or inplants. The use of synthetic gelatins eliminates many of the variablesand drawbacks associated with tissue-derived materials, includingprovoking unwanted immune responses. For example, fish gelatinsdemonstrate high allergenicity and animal gelatins demonstratelow-moderate allergencity, while recombinant gelatins can have zeroallergenicity. In human safety studies, no adverse events related torecombinant gelatin were found.

Methods of creating recombinant gelatins and the benefits of their useare fully described in U.S. Pat. Nos. 6,413,742 and 6,992,172, which arehereby incorporated by reference as if fully set forth herein.

Recombinant gelatins can be produced to be highly (99%) purified.Recombinant gelatin production allows for the optional production ofgelatins with at least one defined and predetermined characteristic,including but not limited to defined molecular weights, pI (isoelectricpoint), guaranteed lot-to-lot reproducibility, and the ability to tailorthe molecule to match a specific application.

An example of tailoring a molecule to match a specific application hasbeen previously described wherein a gelatin was created to be highlyhydrophilic (Werten M W T, et al. (2001). Protein Engineering. 14 (6):447-454). Optionally and preferably a gelatin according to the presentinvention comprises a gelatin having at least one adjusted, tailored orpredetermined characteristic.

Non-limiting examples of other types of characteristics which mayoptionally be so tailored according to the present invention includeundergoing or not undergoing thermoreversible gelation. Recombinantgelatins can be created to undergo thermoreversible gelation or notundergo thermoreversible gelation. A gelatin that has one or morebeneficial characteristics of natural animal gelatin but does notundergo thermoreversible gelation has tremendous amount of utility inenabling the cross-linking of gelatin by other means at temperatures atwhich it would normally undergo thermoreversible gelation. Such agelatin is also encompassed by some embodiments of the presentinvention.

Animal (bovine, porcine and so forth) gelatin, warm water fish gelatin,and recombinant gelatin (gelling type) can undergo thermoreversiblegelation somewhere between 35-40 degrees, particularly at high molecularweights and/or high concentrations (>20%) and/or with modification(s)and/or one or more additional materials (see below for a description).At room temperature, they are in gel form and cannot easily mix withmTG. Various modifications of a composition according to someembodiments of the present invention are described below to maintain thegelatin solutions in liquid form at room temperature.

Cold water fish gelatin and recombinant gelatin (non-gelling type) donot form thermoreversible gels at room temperature, even without furthermodification and/or the presence of one or more additional materials.They have transition points far below room temperature. At roomtemperature, they stay in solution and can react with mTG withoutfurther modification.

According to preferred embodiments of the present invention with regardto recombinant gelatin, a suitable in vitro culturing system is used toproduce the recombinant gelatin. In addition to the use of recombinantmethylotrophic yeast systems for the production of recombinant gelatin,other organisms have been used.

Recombinant, gelatin-like proteins have been expressed in Escherichiacoli though expression levels usually obtained in E. coli are rather lowand purification of the intracellularly produced protein can bedifficult. Bacillus brevis has been used for the expression ofgelatin-like proteins wherein sequence stretches were selected fromnatural collagen genes and polymerized to form semi-synthetic gelatin(Werten M W T, et al. Secreted production of a custom-designed, highlyhydrophilic gelatin in Pichia pastoris. Protein Engineering, Vol. 14,No. 6, 447-454, June 2001).

Additional successful efforts at producing recombinant gelatin haveincluded the production of recombinant gelatin using mammalian andinsect cells. Collagen and gelatin have also been expressed intransgenic tobacco plants, transgenic mice. A transgenic silkworm systemhas been used to produce a fusion protein containing a collagenoussequence. These systems lack sufficient endogenous prolyl hydroxylaseactivity to produce fully hydroxylated collagen, which can be overcomeby over-expression of prolyl hydroxylase (Olsen D, et al. Recombinantcollagen and gelatin for drug delivery. Adv Drug Deliv Rev. 2003November 28; 55(12):1547-67). Plant based systems may also optionally beused; for example a collaboration between Iowa State University andFibrogen is developing the expression of gelatin in transgenic corn.

The gelatin employed in the hemostatic dressing can be a gelatin complexor any gelatin, or a derivative or metabolite thereof, or a gelatinproduced according to a single process or a plurality of processes. Forexample, the gelatin may optionally comprise gelatin type A or gelatintype B, or a combination thereof.

The transglutaminase may optionally comprise any plant, animal, ormicrobe derived transglutaminase, preferably other than blood derivedFactor XIII. Preferably, microbial transglutaminase derived fromStreptoverticillium mobaraensis is used.

The transglutaminase may optionally be in a composition comprising atleast one other substance, such as a stabilizer or filler for example.Non-limiting examples of such materials include maltodextrin, hydrolyzedskim milk protein or any other protein substance, sodium chloride,safflower oil, trisodium phosphate, sodium caseinate or lactose, or acombination thereof.

Although the optimal pH for activity of crude transglutaminase is 6.0,it also functions with high activity in the range of pH 5.0 to pH 8.0.Therefore, a composition according to the present invention forhemostasis preferably has a pH value in a range of from about 5 to about8.

Transglutaminase features a negative temperature coefficient. Over thetemperature range of the transglutaminase activity, it takes a shortertime to react at higher temperatures and longer amount of time to startfunctioning at lower temperatures. The following table shows differentreaction times at different temperatures comparing the same reactiongrade as the reaction at 50° C., pH 6.0 that occurs in 10 minutes:

TABLE 1 reaction temperature of transglutaminase Temperature 5° C. 15°C. 20° C. 30° C. 40° C. Time (minutes) 240 105 70 35 20

Non-limiting examples of commercially available transglutaminaseproducts include those produced by Ajinomoto Co. (Kawasaki, Japan). Apreferred example of such a product from this company is the ActivaTG-TI (In Europe: Activa WM)—Ingredients: mTG and maltodextrin;Activity: 81-135 U/g of Activa. Other non-limiting examples of suitableproducts from this company include Activa TG-FP (ingredients: hydrolyzedskim milk protein, mTG; activity: 34-65 U/g of Activa TG-FP); ActivaTG-GS (ingredients: sodium chloride, gelatin, trisodium phosphate,maltodextrin, mTG, and safflower oil (processing aid); activity: 47-82U/g of Activa TG-GS); Active TG-RM (In Europe: Activa EB)—ingredients:sodium caseinate, maltodextrin, and mTG; activity: 34-65 U/g of Activa;Activa MP (ingredients: mTG, Lactose and Maltodextrin; activity: 78-126U/g of Activa).

Other non-limiting examples of commercially available transglutaminaseproducts include those produced by Yiming Biological Products Co.(Jiangsu, China). A preferred example of such a product from thiscompany is the TG-B (ingredients: 1% mTG, 99% co-protein; activity:80-130 U/g of TG-B). Other non-limiting examples of suitable productsfrom this company include TG-A (ingredients: 0.5% mTG, 99.5% co-protein;activity: 40-65 U/g of TG-A).

For both examples, preferred transglutaminase products are those withthe highest specific activity and simplest co-ingredients, as they arebelieved (without wishing to be limited by a single hypothesis) to havethe best reactivity upon application and a lower potential for undesiredside effects.

In another embodiment, a transglutaminase may optionally be extractedfrom Streptoverticillium Baldaccii or a Streptomyces Hygroscopicusstrain to produce enzyme variants that have been shown to functionoptimally at lower temperatures (approximately 37° C. and 37° C.-45° C.,respectively) (Negus S S. A Novel Microbial Transglutaminase DerivedFrom Streptoverticillium Baldaccii. PhD Thesis. School of Biomolecularand Biomedical Science. Griffith University, Queensland, Australia andCui L et al. Purification and characterization of transglutaminase froma newly isolated Streptomyces hygroscopicus. 2007: 105(2). p. 612-618.).Higher specific activity at lower temperatures is desirable forachieving faster and stronger cross linking of the gelatin under ambientconditions.

According to some embodiments, transglutaminase can be used in the formof any of the above described compositions, optionally including any ofthe commercially available mixtures that include transglutaminase.

In another embodiment, any of the above transglutaminase mixtures mayoptionally be purified by means of gel filtration, cation-exchangechromatography, hollow fiber filtration, or tangential flow filtrationto remove their carrier proteins and/or carbohydrates. Some of thesemethods have been previously described (Bertoni F, Barbani N, Giusti P,Ciardelli G. Transglutaminase reactivity with gelatine: perspectiveapplications in tissue engineering Biotechnol Lett (2006) 28:697-702)(Broderick E P, et al. Enzymatic Stabilization of Gelatin-BasedScaffolds J Biomed Mater Res 72B: 37-42, 2005). The filter pore sizeused for filtration is preferably approximately 10 kDA.

Regardless, the activity of transglutaminase is preferably measuredprior to use and/or manufacture of a composition according to thepresent invention with a transglutaminase reactivity assay. Such anassay may optionally include but is not limited to the HydroxamateMethod, Nessler's Assay, a Colorimetric Assay, or any other assay oftransglutaminase activity (see for example Folk J E, Cole P W.Transglutaminase: mechanistic features of the active site as determinedby kinetic and inhibitor studies. Biochim Biophys Acta. 1966;122:244-64; or the Nessler Assay as described in: Bertoni F, Barbani N,Giusti P, Ciardelli G. Transglutaminase reactivity with gelatine:perspective applications in tissue engineering Biotechnol Lett (2006)28:697-702).

In general, the purity and/or quality of the gelatin and/or thetransglutaminase for use in the hemostatic composition will be of anappropriate purity known to one of ordinary skill in the relevant art tolead to efficacy and stability of the protein.

Proteins for Cross-Linking Substrates Other than Gelatin

As noted above, the cross-linkable protein preferably comprises gelatinbut may also, additionally or alternatively, comprise another type ofprotein. According to some embodiments of the present invention, theprotein is also a substrate for transglutaminase, and preferablyfeatures appropriate transglutaminase-specific polypeptide and polymersequences. These proteins may optionally include but are not limited tosynthesized polymer sequences that independently have the properties toform a bioadhesive or polymers that have been more preferably modifiedwith transglutaminase-specific substrates that enhance the ability ofthe material to be cross-linked by transglutaminase. Non-limitingexamples of each of these types of materials are described below.

Synthesized polypeptide and polymer sequences with an appropriatetransglutaminase target for cross-linking have been developed that havetransition points preferably from about 20 to about 40° C. Preferredphysical characteristics include but are not limited to the ability tobind tissue and the ability to form fibers. Like gelling type gelatins(described above), these polypeptides may optionally be used incompositions that also feature one or more substances that lower theirtransition point.

Non-limiting examples of such peptides are described in U.S. Pat. Nos.5,428,014 and 5,939,385, both filed by ZymoGenetics Inc, both of whichare hereby incorporated by reference as if fully set forth herein. U.S.Pat. No. 5,428,014 describes biocompatible, bioadhesive,transglutaminase cross-linkable polypeptides wherein transglutaminase isknown to catalyze an acyl-transfer reaction between the γ-carboxamidegroup of protein-bound glutaminyl residues and the ε-amino group of Lysresidues, resulting in the formation of ε-(γ-glutamyl)lysine isopeptidebonds.

For example, polypeptides having 13-120 amino acid residues aredescribed, comprising a segment of the formula S1-Y-S2, wherein: S1 isThr-Ile-Gly-Glu-Gly-Gln; Y is a spacer peptide of 1-7 amino acids or notpresent; and S2 is Xaa-Lys-Xaa-Ala-Gly-Asp-Val. Optionally, the spacerpeptide Y is Gln-His-His-Leu-Gly, Gln-His-His-Leu-Gly-Gly orHis-His-Leu-Gly-Gly. Also optionally, Xaa, amino acid 1, of S2 is Ala orSer. Optionally, the spacer peptide comprises His-His-Leu-Gly.Optionally and preferably, at least one of Y and S2 are free of Glnresidues. Optionally, the carboxyl terminal amino acid residue of thepolypeptide is Pro or Gly. Specific non-limiting examples of thepolypeptides include the following:Thr-Ile-Gly-Glu-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val,Thr-Ile-Gly-Glu-Gly-Gln-Gln-His-His-Leu-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val,Thr-Ile-Gly-Glu-Gly-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val,or Leu-Ser-Gln-Ser-Lys-Val-Gly. The patent also describes high molecularweight, biocompatible, bioadhesive, transglutaminase-cross-linkablecopolymers and homopolymers involving these peptides.

U.S. Pat. No. 5,939,385 describes biocompatible, bioadhesivetransglutaminase cross-linkable polypeptides. These polypeptidespreferably have about 9-120 amino acid residues comprising a segment ofthe formula S1-Y-S2, wherein: S1 is selected from the group consistingof Ile-Gly-Glu-Gly-Gln, Gly-Glu-Gly-Gln, Glu-Gly-Gln, and Gly-Gln; Y isHis-His-Leu-Gly-Gly or His-His-Leu-Gly; and S2 is selected from thegroup consisting of Ala-Lys-Gln-Ala-Gly-Asp, Ala-Lys-Gln-Ala-Gly,Ala-Lys-Gln-Ala, Ala-Lys-Gln, Ala-Lys-Ala-Gly-Asp-Val, Ala-Lys-Ala andAla-Lys, wherein said polypeptide has an amino-terminus and acarboxy-terminus and is cross-linkable by a transglutaminase. Apreferred polypeptide is Gly-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln. Alsopreferred is a polypeptide wherein the polypeptide is flanked on eitheror both the amino-terminus and the carboxy-terminus by an elastomericpolypeptide. It further provides an elastomeric polypeptide wherein theelastomeric polypeptide is a pentapeptide or a tetrapeptide,particularly a flanked polypeptide wherein the flanking elastomericpolypeptide is Val-Pro-Gly-Val-Gly, Ala-Pro-Gly-Val-Gly,Gly-Val-Gly-Val-Pro, Val-Pro-Gly-Gly or any portion thereof, preferablysuch that the amino-terminus of the flanked polypeptide is Val and thecarboxy-terminus of the flanked polypeptide is Gly. The patent alsodescribes high molecular weight, biocompatible, bioadhesive,transglutaminase-cross-linkable copolymers and homopolymers involvingthese peptides.

These patents recognize the utility of the described peptides andpolymers for use as tissue adhesives, in wound closure, and in variousother medical applications. However, both patents note that the desiredtransition points of these peptides and polymer are 20-40° C. andrecognize the need to lower the transition point so that thepeptide/polymer will be able to react with transglutaminase in a woundsite. Both patents state: “The transition temperature of the polymer canbe adjusted by the number of polypeptides polypeptide monomers capableof being cross-linked by a transglutaminase. As will be appreciated byone skilled in the art, for clinical applications, reduction of thetransition temperature at the time of application will facilitate therapid solidification of the matrix at the wound site.”

Naturally, in order to ensure the maximal cohesive and adhesive strengthof a cross-linked polymer intended for use as a bioadhesive, it isfrequently not possible to remove cross-linkable monomers. In fact, tomaximize the cohesive and adhesive strengths of such adhesives, it isgenerally preferable to add more cross-linkable monomer substrates.Thus, the bioadhesive potential of the polymers described in thesepatents is significantly limited by the transition temperature of thepolymer solution.

Preferred embodiments of the present invention significantly advance theutility of these polypeptides or polymers for use in hemostatic, tissueadhesive, and tissue sealant applications. For example, optionalembodiments are described below for lowering the transition point ofpolymers that gel at room temperature. These strategies can be utilizedin lowering the transition point of the peptide sequences and polymersdescribed in these patents.

Also as described in greater detail below, preferably the amount ofthese polymers differs for embodiments of the present invention, asopposed to the original uses described in the above patents (whichneither teach nor suggest any of the uses described herein for thepresent invention). For example, the polymer concentration ranges taughtin these patents for use in a tissue adhesive kit are 5 to 100 mg/ml andpreferably 35 to 50 mg/ml. Some embodiments of the present inventioncomprise higher polymer concentrations, for example in the range of150-250 mg/ml. Higher transglutaminase concentrations are also preferredfor some embodiments of the present invention.

Other synthetic substrates may also optionally be provided according tosome embodiments of the present invention. Preferably shorttransglutaminase substrates are synthesized and then connected and/orbound to large polymer molecules. The transglutaminase substrates aregenerally very short (<20 amino acid residues). The solubility andtransition point of such substrates is dependent on the polymer to whichthe substrate is attached. For example, if the substrate is attached togelling gelatin, then a solution of this newly synthesize molecule wouldrequire the addition of another substance to stay in liquid form at roomtemperature.

A non-limiting example of this type of material is described in U.S.Pat. No. 7,208,171, which is hereby incorporated by reference as iffully set forth herein, which describes the rational design oftransglutaminase substrate peptides. The design strategy was based onmaximizing the number of available acyl acceptor lysine-peptidesubstrates and acyl donor glutaminyl-peptide substrates available fortransglutaminase cross-linking. Beyond this, the Lys and Glu substratepeptides were designed to possess basic features of knownbiomacromolecular and synthetic peptide substrates of transglutaminase.For example, the Glu substrate peptides contained 2-5 contiguous Gluresidues, based on evidence that peptides become better transglutaminasesubstrates with increasing length of Glu repeats (Gorman, J. J.; Folk,J. E. J. Biol. Chem. 1980, 255, 419-427. & Kahlem, P.; Terre, C.; Green,H.; Djian, P. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14580-14585.) andthat proteins containing two or more adjacent Glu residues are known tobe good substrates. (Etoh, Y.; Simon, M.; Green, H. Biochem. Biophys.Res. Commun 1986, 136, 51-56. & Hohenadl, C.; Mann, K.; Mayer, U.;Timpl, R.; Paulsson, R.; Aeschlimann, D. J. Biol. Chem. 1995, 270,23415-23420.) A Leu residue was placed adjacent to the Glu near theC-terminus in several peptides, because this has been shown to result ina significant increase in Glu specificity. (Gross, M.; Whetzel, N. K.;Folk, J. E. J. Biol. Chem. 1975, 250, 4648-4655.) Regarding the Lyssubstrate peptides, it has been shown that the composition and sequenceof the amino acids adjacent to lysine residues in peptide and proteinsubstrates can have an effect on the amine specificity. (Groenen, P.;Smulders, R.; Peters, R. F. R.; Grootjans, J. J.; Vandenijssel, P.;Bloemendal, H.; Dejong, W. W. Eur. J. Biochem. 1994, 220, 795-799. &Grootjans, J. J.; Groenen, P.; Dejong, W. W. J. Biol. Chem. 1995, 270,22855-22858.). Finally, in all peptides a Gly residue was added on theC-terminal side to act as a spacer between the peptide and the polymerin the peptide-polymer conjugates, so that the peptide in the conjugatemay be more accessible to enzyme.

Transglutaminase specificity assays of the peptides described in thispatent demonstrated that they successfully created acyl acceptor andacyl donor substrates with high transglutaminase binding specificity. Itis suggested in the patent that these substrates can be covalentlyconjugated to PEG, dendrimers, chitosan, gelatin, soluble collagens,hyaluronic acid, alginates, and albumins. The patent goes on to suggestthat such polymer-peptide conjugates in solution or liquid form could beused a surgical sealants and/or medical adhesives.

Although this patent describes highly specifictransglutaminase-cross-linkable peptide substrates, it does not teach orsuggest the advanced application methods or material modificationsdescribed as part of the present invention, nor does it teach or suggestthe compositions of the present invention. The taught substrates of thepatent however, may optionally be useful in enhancing a bioadhesivecreated through transglutaminase cross-linking or in creating such abioadhesive from an otherwise non-transglutaminase-specific polymer.These substrates would need to be combined with one or more otherproteins or scaffolds as described herein to be useful for the presentinvention.

Cross-Linking Materials Other than Transglutaminase

As noted above, the cross-linking material preferably comprisestransglutaminase but may also, additionally or alternatively, compriseanother type of cross-linking material.

Non-limiting examples of such cross-linking agents include carbodiimidessuch as N,N-(3-(dimethylamino)propyl)-N-ethyl carbodiimide (EDC),N-hydroxysuccinimide (NHS) with EDC, or carbodiimides used together withpoly(L-glutamic acid) (PLGA) and polyacrylic acid. In anotherembodiment, such cross-linking agents can include Tyrosinase orTyrosinase with chitosan. In another embodiment, cross-linking(polymerization) is photo-initiated with ultraviolet light or γ-rays. Inanother embodiment, cross-linking agents can include alkylene, citricacid (carbonic acid), or Nano-hydroxyapataite (n-HA)+poly(vinyl alcohol)(PVA).

In another embodiment, a cross-linking agent is a plant-derivedpolyphenol such as (i.e. hydroxylated cinnamic acids, such as caffeicacid (3,4-dihydroxycinnamic acid), chlorogenic acid (its quinic acidester), caftaric acid (its tartaric acid ester), and flavonoids (i.e. asquercetin and rutin). In another embodiment, the additionalcross-linking agent is an oxidized mono or disaccharide, oxo-lactose, ora dialdehyde based on a sugar moiety (galacto-hexodialdose) (GALA). Inanother embodiment, Genipin or other iridoid glycoside derivative, orSecoiridoids, preferable oleuropein, comprises the cross-linking agent.In another embodiment, the cross-linking agent is a thiol-reactivepoly(ethylene glycol). In another embodiment, the cross-linking agent isdextran, oxidized dextran, dextran dialdehyde. In another embodiment,the cross-linking agent is a multi-copper oxidase such as laccase orbilirubin oxidase.

Illustrative Compositions

The above described cross-linking substrates and cross-linking materialsmay optionally be combined with one or more additional materials to formvarious compositions according to the present invention. According tosome embodiments, the adhesive material optionally and preferablycomprises: (i) gelatin; (ii) a transglutaminase; wherein the gelatin andtransglutaminase are formed into particles either separately ortogether. More preferably, the gelatin and transglutaminase are providedin sufficient quantities to be useful as a sealing, hemostatic agent.

Various amounts of each and their ratios were previously described. Thetransglutaminase content may optionally be increased to increase therate of reaction or decreased to enhance safety. According to someembodiments of the present invention, a 15-30% solution of gelatin ispreferably applied, followed by a 15-30% solution of transglutaminase.

In addition, one or more supplements can also be contained in thehemostatic product, e.g., drugs such as growth factors, polyclonal andmonoclonal antibodies and other compounds. Illustrative examples of suchsupplements include, but are not limited to: antibiotics, such astetracycline and ciprofloxacin, amoxicillin, and metronidazole;anticoagulants, such as activated protein C, heparin, prostracyclin(PGI2), prostaglandins, leukotrienes, antitransglutaminase III, ADPase,and plasminogen activator; steroids, such as dexamethasone, inhibitorsof prostacyclin, prostaglandins, leukotrienes and/or kinins to inhibitinflammation; cardiovascular drugs, such as calcium channel blockers,vasodilators and vasoconstrictors; chemoattractants; local anestheticssuch as bupivacaine; and antiproliferative/antitumor drugs such as5-fluorouracil (5-FU), taxol and/or taxotere; antivirals, such asgangcyclovir, zidovudine, amantidine, vidarabine, ribaravin,trifluridine, acyclovir, dideoxyuridine and antibodies to viralcomponents or gene products; cytokines, such as alpha- or beta- orgamma-Interferon, alpha- or beta-tumor necrosis factor, andinterleukins; colony stimulating factors; erythropoietin; antifungals,such as diflucan, ketaconizole and nystatin; antiparasitic agents, suchas pentamidine; anti-inflammatory agents, such as alpha-1-anti-trypsinand alpha-1-antichymotrypsin; anesthetics, such as bupivacaine;analgesics; antiseptics; and hormones. Other illustrative supplementsinclude, but are not limited to: vitamins and other nutritionalsupplements; glycoproteins; fibronectin; peptides and proteins;carbohydrates (both simple and/or complex); proteoglycans;antiangiogenins; antigens; lipids or liposomes; and oligonucleotides(sense and/or antisense DNA and/or RNA).

According to some preferred embodiments of the present invention, thereis provided a composition which features gelatin that undergoesthermoreversible cross-linking (as described above, some but not alltypes of gelatin undergo thermoreversible cross-linking withoutmodification and/or the use of one or more additional materials).Thermoreversible gelation of animal gelatin occurs when a gelatinsolution is cooled below approximately body temperature (37° C.). Ingelatin-mTG mixtures at room temperature, this gelation traps the mTG ina thermoreversible gel and prevents it from reacting with the gelatin toform an irreversible sealing gel. As operating room temperature isgenerally maintained at 22° C., thermoreversible gelation of a gelatinsolution will occur rather rapidly in a clinical setting if it is notcontinuously heated. This presents a problem in the application of agelatin-mTG mixture for hemostasis since the gelatin solution has to beheated prior to its mixture with mTG. Having to heat the gelatinimmediately prior to application is undesirable from both a logisticaland safety standpoint as a heating element would need to be added to thesensitive operating room environment for example; in emergencysituations and/or urgent medical care situations outside of operatingrooms, such a requirement for heating is even more problematic.

Aside from its hindrance to tissue adhesion and hemostasis, theinability of gelatin to form a solution at room temperature and mix withmicrobial transglutaminase also presents difficulties for otherpotential applications of the gelatin-mTG mixture. For example, whilegelatin-mTG gels have been used as scaffolds for cell encapsulation intissue engineering, extreme care has had to be taken to ensure that thegelatin solution had sufficiently cooled prior to encapsulation of thecells. Furthermore, implantation of encapsulated cells is quitecomplicated as thermoreversible gelation of the gelatin occurs beforethe gelatin-mTG mixture can be safely implanted in the body. A similarproblem exists with regard to local drug delivery wherein the efficacyof certain drugs could be harmed by coming into contact with heatedgelatin and implantation of a gelatin-mTG gel that incorporates acertain drug could be hindered by the thermoreversible gelation ofgelatin.

Fortunately, cross-linking of gelatin by mTG occurs by linking the Lysand Gln amino groups (Chen et al. Biomacromolecules, Vol. 4, No. 6,2003) whereas the amino acid groups in gelatin that are responsible forits thermoreversible gelation are Pro & Hyp (Haug et al. FoodHydrocolloids 18 (2004) 203-213). Thus, the potential exists forreducing the proclivity of gelatin for thermoreversible gelation withoutharming its ability to form cross-linked gels through mTG cross-linking.In other words, the solubility of the gelatin used in a gelatin-mTGmixture can be increased and its melting point lowered to allow it toform a room temperature solution with mTG without negatively affectingthe cross-linked gelatin-mTG gel that is formed.

According to some embodiments of the present invention, there areprovided compositions of matter of a gelatin-mTG mixture wherein themixture is modified by one of a number of methods to increase thesolubility of the gelatin and allow the gelatin to form a solution withthe mTG at temperatures lower than the natural melting point of standardanimal gelatin. These compositions include (i) gelatin-mTG mixtures madeusing standard gelatin that has been modified to reduce its meltingpoint; (ii) gelatin-mTG mixtures that include additives that increasethe solubility of gelatin in the gelatin-mTG solution itself; (iii)gelatin-mTG mixtures made using commercially available gelatin productstreated to have lower transition temperatures; and (iv) gelatin-mTGmixtures that form a solution under specific, carefully controlledenvironmental conditions (temperature, pH, ionic concentration, etc)that lower the melting point of the gelatin.

These novel compositions greatly increase the utility of gelatin-mTGgels and enable a wide variety of applications, particularly in themedical field, that can utilize gelatin-mTG gels that can be formed bymixing gelatin at mTG at room temperature. In addition, in many cases,the gelatin and gelatin-mTG solutions formed using gelatin solutionsthat contain lower melting point gelatin will have the additionalbenefit of lowering the initial viscosity of the solution, allowing themTG more freedom of movement and increasing the speed at which thegelatin-mTG reaction occurs.

According to some embodiments of the present invention there are alsoprovided additional enhancements to the gelatin-mTG mixture that havethe potential to improve the properties of a gelatin-mTG based product.For example, the present invention also features methods of furtherstabilizing the mTG in the gelatin-mTG mixture to increase its shelflife.

In another embodiment of the invention, a plasticizer is incorporatedinto the gelatin-mTG solution. Plasticizers have been shown to lower themelting point of gelatin, allowing it to form a solution at lowertemperatures without undergoing thermoreversible gelation. One or moreplasticizers are preferentially added to gelatin granules or to agelatin solution to lower its melting point prior to the mixture of thegelatin solution with mTG or mTG solution. In a situation where thegelatin and mTG solutions are lyophilized, one or more plasticizers areadded to the gelatin solution prior to its lyophilization. In analternate embodiment, as described above, one or more plasticizers isadded to the gelatin solution, allowing for the addition of mTG at alower temperature, at which mTG is not highly reactive. Thegelatin-mTG-plasticizer solution can then be lyophilized or otherwisedried in an already mixed form.

In a preferred embodiment, a polyhydric alchohol, or polyol, is used asthe plasticizer. Such polyols include glycerine, glycerol, xylitol,sucrose, and sorbitol. Sorbitol makes gelatin-mTG gels more elastic andsticky. Glycerol makes gelatin-mTG gels stiffer and accelerates mTGcross-linking of gelatin. A preferred concentration ratio range forglycerol is preferably from about 0.5:1 to about 5:1 Glycerol:Gelatin,more preferably from about 1:1 to about 2:1 Glycerol:Gelatin, weight perweight. A preferred concentration ratio range for sorbitol is preferablyfrom about 0.5:1 to about 5:1 Sorbitol:Gelatin, more preferably fromabout 1:1 to about 3:1 Sorbitol:Gelatin, weight per weight.

Polyhydric alcohols have higher boiling points when compared to similarsized monohydric alcohols. The water solubilities of polyhydric alcoholsare higher when compared to similar sized monohydric alcohols sincethere are more hydroxyl groups for water molecules to be attracted to.In the food industry, polyhydric alcohols such as glycerine are used toincrease the water solubility of gelatin as described in U.S. Pat. No.2,558,065, hereby incorporated by reference as if fully set forthherein, where a polyhydric alchohol such as glycerine is poured overgelatin granules, and U.S. Pat. No. 3,939,001, hereby incorporated byreference as if fully set forth herein, where gelatin is allowed toabsorb the polyhydric alchohol for a period of time sufficient for thegelatin granules to become swollen but prior to dissolution. Thesetechniques and the variations of these techniques described in thosepatents are to be considered preferential embodiments of the polyolusage described as part of the current invention.

The effects of different concentrations of the plasticizers glycerol,xylitol, sorbitol, sucrose, and trehalose on lowering the transitionpoints of gelatin are well documented (D'Cruz N M, Bell L N. ThermalUnfolding of Gelatin in Solids as Affected by the Glass Transition. JFood Science 2005: 70(2), Kozlov P V, Burdygina G I. The structure andproperties of solid gelatin and the principles of their modification.Polymer, 1983 (24): p. 651-666).

While the effect of polyhydric alcohols on the melting point of gelatinhas been well documented, they have never been used, prior to thecurrent invention, with gelatin or a gelatin solution prior to itsmixture with mTG or with a gelatin-mTG solution.

In an embodiment of the addition of polyol plasticizers, a preferredrange for the weight ratio of plasticizer to gelatin is preferably fromabout 0.5:1 to about 1:1, plasticizer:gelatin.

In another embodiment of using gelatin plasticizers to lower the meltingpoint of gelatin in solution, the type of plasticizer used can includetriethanolamine, resorcin, thiodiglycol, sodium salt oftoluenesulphoacid, butylene glycol, urea nitrate, thiourea, urea,glutamic acid, aspargic acid, valine, glycine, KSCN, KI, and LiBr.

The addition of urea to gelatin solution has been previously exploredand demonstrated the ability to prevent high molecular weight gelatin(99 kDa) from forming a thermoreversible gel at 25° C. (Otani Y, TabataY, &a& Y. Effect of additives on gelation and tissue adhesion ofgelatin-poly(L-glutamic acid) mixture. Biomaterials 19 (1998) 2167-2173)

In a preferred embodiment of using urea to prevent thermoreversiblegelation in gelatin or gelatin-mTG solution at temperatures below theirnatural melting points, urea is added to the solutions at a urea:gelatinratio between 0.25-2.0, weight per weight. Even more preferentially,urea is added to the solutions at a urea:gelatin ratio of from about 1:2to about 2:2, weight per weight.

In another embodiment of the present invention, the pH level and ionconcentration of an aqueous solvent are modified to increase thesolubility of gelatin dissolved in the solvent. The further the productpH is from the isoionic pH the better will be the solubility of thegelatin. A preferred aqueous solvent used in this technique is phosphatebuffered saline (PBS). Other suitable buffers include borate, phosphate,HEPES (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic sacid]) and thelike.

Generally, in compositions that are to be used within living organisms,it is preferred to dissolve gelatin in an aqueous solvent buffered at pH5.5-9.0 and of low to moderate ionic strength (equivalent to about 1 to1000 mM NaCl, preferably 100 to 150 mM NaCl) More preferably, the pH ofthe solution is about 6.0-8.0, more preferably about 7.4. Though gelatinis soluble at these pH and ion concentrations, its solubility can beincreased by increasing the disparity between the solution pH and theisoionic pH of gelatin.

One or more salts may also optionally be added to lower the transitiontemperature of gelatin. Preferably the salts are added at a suitableconcentration range for reducing the transition temperature, morepreferably below room temperature. For example, the following salts atthe indicated concentration ranges were found to reduce the transitionpoint of gelatin below room temperature: Sodium Bromide (1-2 M), SodiumNitrate(1-2 M), Sodium Thiocyanate (0.5-1.5 M), Sodium Iodide(0.5-1.5M), Sodium Benzenesulfonate(0.5-1.5 M), Sodium Salicylate(0.25-1 M),Sodium Dichloroacetate (1-2 M), Sodium Trichloroacetate(0.5-1.5 M),Sodium Dibromoacetate(0.5-1.5 M), Sodium Tribromoacetate(0.25-1 M),Sodium Diiodoacetate (0.5-1.5 M), Sodium Acetyltryptophan (0.5-1.5 M),Sodium Acetylenedicarboxylate (1-2 M), Lithium Salicylate(1-2 M),Lithium Diidodosalicylate(0.2-1 M) (see for example Bello J, Riese H CA, Vinograd J R. Mechanism of Gelation of Gelatin. Influence of CertainElectrolytes on the Melting Points of Gels of Gelatin and ChemicallyModified Gelatins. Am Chem Soc. September 1956 (60). P. 1299-1306).

Optionally and preferably, one or more acidic substances are added tothe composition to decrease the pH. Lowering the pH of the gelatinsolution reduces its transition point. In general, reducing the gelatintransition point is useful to reconstitute the gelatin in the body at 37degrees. For some preferred types of gelatin used herein, as for examplefrom mammalian sources, lowering the pH provides better results for thegelatin transition point. However, for some types of gelatin, raisingthe pH may optionally provide better results.

In another embodiment of increasing the pH disparity between the gelatinsolution and the isoionic point of gelatin, the gelatin itself ismodified. This can be accomplished by treating it prior to dissolving itin solution to create an electrostatically charged gelatin, as in U.S.Pat. No. 6,863,783, hereby incorporated by reference as if fully setforth herein, or by controlling the isoelectric point of gelatin, as inU.S. Pat. No. 2,398,004, hereby incorporated by reference as if fullyset forth herein.

If the pH of the gelatin solution is raised, instead of lowered, thenthe pH of the solution will be closer to the isoionic point of gelatinand the transition point will be raised. This modification couldoptionally be used to maintain non-cross-linked gelatin as athermoreversible gel after implantation in the body.

In another embodiment of the current invention, the gelatin, mTG, orboth substances have undergone drying after being mixed with a trehalosecarbohydrate or other carbohydrate to stabilize the protein or enzyme inits active form, enabling it to be reconstituted with ease. Variousembodiments of drying are lyophilization, spray drying, drum drying, airdrying, heated drying, vacuum drying, or any other method of drying agelatin-trehalose or gelatin-mTG-trehalose solution.

The superior stabilizing ability of trehalose in air drying and freezedrying has been well documented. It has been shown that dried materialsundergo quicker reconstitution when drying is performed after trehalosehas been added to a particular material or solution (Crowe L M, Reid DS, Crowe J H. Is Trehalose Special for Preserving Dry Biomaterials?Biophysical Journal 1996 (71): 2087-2093).

The drying of protein solutions incorporating trehalose at ambienttemperature and atmospheric pressure has been fully described in U.S.Pat. No. 4,891,319. In the examples described therein, the function ofthe dried proteins was preserved.

In the specific case of gelatin, the incorporation of trehalose into thegelatin solution has the additional benefit of increasing the strengthof gelatin gels. (Norie N, Kazuhiro M, Masami N, Yusuke O, Takashi O,Keiko N. Factors Affecting the Gelation of a Gelatin Solution in thePresence of Sugar. Journal of Home Economics of Japan. 55(2): p. 159-166(2004))

Furthermore, the gelatin-mTG cross-linking reaction bears manysimilarities to the cross-linking reaction of natural blood factors.Trehalose drying to stabilize blood factors has recently beendemonstrated (U.S. Pat. Nos. 6,649,386 and 7,220,836) and is in theprocess of being used commercially to prepare products with bloodproteins that can be easily reconstituted (ProFibrix™, Leiderdorp).

In another embodiment of the current invention, gelatin is dried in thepresence of a sugar. This can include the spray-atomizing of gelatins ondifferent supports such as sugar, maltodextrins, or starches.

In an optional embodiment of the current invention, a commercial gelatinproduct called Cryogel™ produced by PB Gelatins (Tessenderlo Group,Belgium) is used. Cryogel is soluble at temperatures 5-6° C. less thanequivalent untreated gelatin. The precise treatment process used in theproduction of Cryogel is proprietary.

In yet another embodiment of the present invention, the composition mayoptionally feature one or more additional substances. For example, thecomposition may optionally comprise a denaturant, including but notlimited to one or more of Guanidine Hydrochloride, or Urea. Thecomposition may also optionally, alternatively or additionally, comprisea reducing agent, including but not limited to one or more of magnesiumchloride or hydroquinone. The composition may also optionally,alternatively or additionally, comprise a substance such as isopropanolfor increasing hydrogen bond formation. The composition may alsooptionally, alternatively or additionally, comprise a protic polarsolvent, preferably being capable of structurally interacting withproteins and preventing helix formation in gelatin, such as DMSO(dimethylsulfoxide). The composition may also optionally, alternativelyor additionally, comprise a desiccant which is exothermic upon enteringsolution, such as calcium chloride for example.

According to some embodiments, the present invention also features agelatin specific protease which comprises an enzyme or enzyme mixturethat can rapidly breakdown gelatin molecule strands but does notadversely affect the natural fibrin-based clotting networks.

A gelatin-specific protease could optionally be used to remove thebandage/dressing/absorbable hemostat/sealant from a wound site withoutdamaging the natural endogenous fibrin clot and causing rebleeding. Thisfeature is an additional benefit of the present invention as compared toexisting products, and solves the technical problem that hemostaticdressings which are sufficiently adhesive to adhere well to a wound siteand stop bleeding cannot be removed without removing or destroying thefibrin clot. Although at least some embodiments of a bandage accordingto the present invention are reabsorbable, there may be a need to removeit from a wound if the doctor wants to operate on a wound site, or put abandage in a different place.

An exemplary non-limiting protease is proteinase K (Chen, et al.Biomacromolecules 2003, 4, 1558-1563). However, other proteases,particularly quicker acting enzymes, may be used alternatively.

According to other embodiments, one or more protease inhibitors areoptionally added, including but not limited to aprotinin, transexamicacid, alpha-2 plasmin inhibitor, alpha-1 antitrypsin, or the Pittsburghmutant of alpha-1-antitrypsin (Arg-358 alpha-1-antitrypsin; see Owen etal. N. Engl. J. Med. 309: 694-698, 1983 and U.S. Pat. No. 4,711,848,which is hereby incorporated by reference as if fully set forth herein).Within a preferred embodiment, aprotinin is included in an amountsufficient to provide a final working concentration of 1500-20,000KIU/mL.

According to other embodiments, the hemostatic material of the presentinvention may further comprise an additional hemostatic substance, inaddition to gelatin and TG. Such a substance can be biological orsynthetic in nature and can include but is not limited to one or moreknown hemostatic agents such as albumin, collagen, fibrin, thrombin,chitosan, ferric sulfate, or other metal sulfates.

According to still other embodiments, the hemostatic material of thepresent invention may further comprise an accelerant for acceleratingthe rate of cross-linking upon combining the cross-linking material,such as transglutaminase for example, and gelatin. Such an accelerantmay optionally comprise calcium for example.

Calcium is a preferred component of the transglutaminase/gelatincross-linking reaction. Various studies have demonstrated that varyingcalcium concentrations and/or the addition of calcium-mobilizing drugs(including but not limited to maitotoxin (MTX)) can speed up thetransglutaminase clotting reaction. Therefore, according to embodimentsof the present invention, calcium and/or calcium-mobilizing drugs areincluded, although alternatively no calcium and/or calcium-mobilizingdrug is used. These modifications for using calcium are useful withcalcium-dependant transglutaminases but not with calcium-independenttransglutaminases.

According to still other embodiments, the hemostatic material of thepresent invention may further comprise a material for inducing anexothermic reaction, preferably upon combining the cross-linkingmaterial, such as transglutaminase for example, and gelatin. Theinduction of an exothermic reaction may optionally and preferablysupport cross-linking even under conditions of an ambient environment,wherein “ambient” may optionally be defined as any environment having atemperature of less than about 30° C. Such an exothermic agent mayoptionally comprise one or more of calcium, chlorine containingmolecules (such as calcium chloride or magnesium chloride), or metallicoxides/zeolites for example, or a combination thereof.

Composition Preparation

Compositions as described herein may optionally be prepared according toone or more different methods of various embodiments of the presentinvention. In one embodiment of the invention, gelatin in thegelatin-mTG mixture is subjected to very specific drying methods thatinvolve the use of heat prior to its mixture with the mTG. These dryingmethods increase the solubility of gelatin by lowering its meltingpoint, preferably below operating room temperatures. The drying methodscan increase the solubility of gelatin without any additives and withoutaltering the environmental conditions under which gelatin or gelatin-mTGsolutions are formed. Nonetheless, the addition of certain additives,such as plasticizers or stabilizers, or the manipulation of certainenvironmental factors, such as temperature, ion concentration, andosmotic pressure, of the gelatin or gelatin-mTG solutions may be used tofurther enhance the properties of a gelatin-mTG mixture that alreadyincorporates gelatin dried using a technique that reduces its meltingpoint.

In a preferred embodiment of heat-dependant gelatin drying to prepare agelatin that can form a solution with mTG at reduced temperatures, apure gelatin solution having a water content of at least 35% is sprayedat a temperature in excess of the gelation and solidificationtemperature on an excess of finely divided solid gelatin particles whichcontain less than 8% of water. The particles are then dried in a fluidbed to a water content of from 8 to 13%. This process, as well asvariations of this process that are also encompassed within the scope ofthe present invention, are described in detail in U.S. Pat. No.4,889,920, hereby incorporated by reference as if fully set forthherein.

In another preferred embodiment of heat-dependant gelatin drying toprepare a gelatin that can form a solution with mTG at reducedtemperatures, a gelatin, having a water content of more than 8% byweight based on the total weight of the gelatin and water, is subjectedto microwave heating to remove at least 35% of said water content toobtain a treated gelatin having a water content of not more than 16% ofweight based on the total weight of the gelatin and water. This process,as well as variations of this process that should also be consideredpart of the current invention, are described in detail in U.S. Pat. No.4,224,348, hereby incorporated by reference as if fully set forthherein.

In another embodiment of heat-dependant gelatin drying to prepare agelatin that can form a solution with mTG at reduced temperatures,gelatin is dried at 100° C. under reduced pressure as described in U.S.Pat. No. 2,803,548, hereby incorporated by reference as if fully setforth herein. This process alters the gelatin strands themselves,rendering them incapable of being thermoreversibly gelled. Though mTGcross-linking of gelatin is not dependant on gelatin's ability to form athermoreversible gel, this drying process results in a weakening of thegelatin strands, and thus of any gel created using such gelatin ingelatin-mTG cross-linking.

In another embodiment of the invention, gelatin in the gelatin-mTGmixture is subjected to very specific drying methods that involve theuse of lyophilization prior to its mixture with the mTG. These dryingmethods increase the solubility of gelatin by lowering its meltingpoint, preferably below operating room temperatures. The drying methodscan increase gelatin's solubility without any additives and withoutaltering the environmental conditions under which gelatin or gelatin-mTGsolutions are formed. Nonetheless, the addition of certain additives,such as plasticizers or stabilizers, or the manipulation of certainenvironmental factors, such as temperature, ion concentration, andosmotic pressure, of the gelatin or gelatin-mTG solutions may be used tofurther enhance the properties of a gelatin-mTG mixture that alreadyincorporates gelatin dried using a lyophilization technique that reducesits melting point.

In a preferred embodiment of lyophilizing gelatin drying to prepare agelatin that can form a solution with mTG at reduced temperatures, agelatin dissolved in water at a concentration of 0.1-2% by weight isfreeze-dried under reduced pressure. This process, as well as variationsof this process that should also be considered part of the currentinvention, are described in detail in U.S. Pat. No. 2,166,074, herebyincorporated by reference as if fully set forth herein.

In another embodiment of the invention, the gelatin-mTG mixture issubjected to lyophilization once the gelatin and mTG have already beenmixed in solution. This lowers the melting point of gelatin whileresulting in an evenly mixed, lyophilized gelatin-mTG mixture where thegelatin in dry form is in contact with the mTG in dry form. In thisembodiment, the gelatin and mTG are simultaneously reconstituted fromlyophilized state and immediately form a solution at the site ofreconstitution. This technique can preferentially be used with gelatinor a gelatin mixture that already has a lower melting point thanstandard gelatin since the activity of mTG decreases exponentially atlower temperatures (below about 37° C.).

Thus, a solution consisting of reduced-melting point gelatin and mTG canbe formed at a low temperature without rapid cross-linking and gelationoccurring. This solution can then be lyophilized, resulting in a driedmixture of homogenously distributed gelatin and mTG. Such a mixture canbe rapidly reconstituted to form a gel when put in contact with a warmersolvent. Such a technique could preferentially be used in a wounddressing, where bodily fluids at their natural temperature of 37° C. canreconstitute the gelatin and mTG.

Preferably, according to some embodiments of the present invention,there is provided a gelatin-mTG particle mixture for hemostatic ortissue sealant purposes wherein the gelatin and mTG are spray driedtogether to create a well dispersed powder containing gelatin and mTG inconcentration appropriate for the hemostatic or tissue sealantapplications.

In another embodiment of the current invention, the gelatin used as partof the gelatin-mTG mixture has been hydrolyzed, partially hydrolyzed, orsome percentage of a gelatin mixture has been hydrolyzed or partiallyhydrolyzed in order to increase its solubility. An example of such atechnique has successfully been demonstrated in a process involving thecoating of standard gelatin granules with a film of hydrolyzed gelatin(U.S. Pat. No. 4,729,897, hereby incorporated by reference as if fullyset forth herein). This embodiment can include the use of gelatin thathas been hydrolyzed or partially hydrolyzed in the presence of aplasticizer, which can include a polyhydric alcohol, a carbohydrate, orother plasticizer as described above.

In another embodiment of the current invention, a solution containingpre-mixed mTG and gelatin, or other protein hydrolysates, is freezedried to increase the stability of the compound. This technique, as usedin preparing a composition used for food processing, is described inU.S. Pat. No. 6,030,821, hereby incorporated by reference as if fullyset forth herein.

In another embodiment of the current invention, the gelatin used as partof the gelatin-mTG mixture is spray dried after being mixed with acid toform a dilutely acidic gelatin solution wherein the acid is kept at5-20% of the level of gelatin, allowing fine droplets to form forenhanced drying. This process is described in Canadian Patent 896,965,hereby incorporated by reference as if fully set forth herein.

In another embodiment of the current invention, one or more of theabove-described techniques for enhancing a product containing gelatinand mTG are used in unison or in series. This can preferentially includeusing two or more plasticizers together in a gelatin or gelatin-mTGsolution, using one or more plasticizers in a gelatin or gelatin-mTGsolution prior to drying it using one of the described drying methods.It can also include drying the gelatin or gelatin-mTG using one dryingtechnique, dissolving the dried gelatin or gelatin-mTG in solution, andthen re-drying the gelatin or gelatin-mTG.

These methods could also optionally be used for composition whichundergoes thermoreversible gelation, preferably including forcompositions comprising various combinations of non gelatin proteins andalso optionally other cross-linkers (other than transglutaminase) forexample.

Bandages

An exemplary embodiment of the present invention is directed to ahemostatic dressing, e.g., for treating wounded tissue in a patient,which comprises gelatin and transglutaminase, preferably separated untiltheir interaction is required or desired for the activity of thebandage. The bandage may optionally feature a non-absorbent backing,such as a plastic backing. The bandage may also optionally feature aresorbable material layer.

Another embodiment of the present invention is directed to a hemostaticdressing for treating wounded tissue in a patient which optionally andpreferably comprises: (i) a gelatin layer; (ii) a transglutaminase layeradjacent to said gelatin layer; wherein the transglutaminase layer iscoextensive or noncoextensive with the gelatin layer.

Another embodiment of the present invention is directed to a hemostaticdressing for treating wounded tissue in a patient which optionally andpreferably comprises: (i) a resorbable or non-resorbable material layer;(ii) a gelatin layer adjacent to said material layer; (iii) atransglutaminase layer adjacent to said gelatin layer; wherein thetransglutaminase layer is coextensive or noncoextensive with the gelatinlayer.

Another embodiment of the present invention is directed to a hemostaticdressing for treating wounded tissue in a patient which comprises: (i) afirst gelatin layer; (ii) a resorbable material layer adjacent to thefirst gelatin layer; (iii) a transglutaminase layer adjacent to theresorbable material layer; and (iv) a second gelatin layer adjacent tothe transglutaminase layer, wherein the transglutaminase layer isnoncoextensive with the first and/or second gelatin layers.

According to some embodiments, the present invention provides ahemostatic dressing (e.g., a bandage) that includes a layer oftransglutaminase sandwiched between a first and a second layer ofgelatin, wherein the transglutaminase layer may be coextensive ornoncoextensive with the first and/or second gelatin layer. Such ahemostatic dressing is useful for treating wounds. The noncoextensivemodel offers the advantage of inhibiting delamination of the layers, ascompared with dressings in which the transglutaminase layer iscoextensive with the entire first and second gelatin layers. However,hemostatic performance of the coextensive model may be superior to thatof the noncoextensive model.

According to other embodiments of the present invention, there isprovided a dressing of the invention which optionally and preferablycomprises: (i) a resorbable or non-resorbable matrix; (ii) gelatin;(iii) a transglutaminase; wherein the gelatin and transglutaminase areincorporated within said matrix.

In another embodiment, the hemostatic device comprises: (i) a porousresorbable or non-resorbable matrix; (ii) gelatin; (iii) atransglutaminase; wherein the gelatin and transglutaminase are adheredto said matrix.

In various embodiments, the transglutaminase layer can be configured inany of a variety of shapes and patterns. For example, and withoutlimitation, the transglutaminase layer can be configured as an array ofspots comprising transglutaminase, or as a single spot comprisingtransglutaminase. Alternatively, the transglutaminase layer can beconfigured as a plurality of lines comprising transglutaminase.

Each layer of the hemostatic dressings can also optionally contain oneor more suitable fillers, binding agents and/or solubilizing agents. Inaddition, each of the hemostatic dressings can also optionally furthercomprise a release layer which contains a release agent and/or a backingmaterial.

According to preferred embodiments, each layer of the hemostaticdressings may optionally contain one or more suitable fillers, such assucrose. Each layer of the hemostatic dressings can also optionallycontain one or more suitable binding agents, such as sucrose. Each ofthe hemostatic dressings can also optionally further comprise a releaselayer which contains a release agent. An exemplary release agent issucrose. Each layer of the hemostatic dressings can also optionallycontain one or more suitable solubilizing agents, such as sucrose.

Without wishing to be limited by a single hypothesis, the properties ofsucrose as part of the present invention may optionally be at leastpartially determined by an amount added. In relatively highconcentrations (20-30% sucrose solution) it can be sprayed onto surface(such as a bandage) to prepare that surface for application of anothersolution (such as gelatin or mTG solution) to be adhered. At lowerconcentrations (around 2% for example), the sucrose can be added to thegelatin or mTG solution to help such a solution adhere to a surface(such as the bandage).

Each layer of the hemostatic dressings can also optionally contain oneor more suitable foaming agents, such as a mixture of citric acid andsodium bicarbonate.

Each of the hemostatic dressings can also further comprise a backingmaterial on the side of the dressing opposite the wound-facing side whenthe dressing is in use. The backing material can be affixed with aphysiologically-acceptable adhesive or can be self-adhering (e.g. byhaving a surface static charge). The backing material can be aresorbable material or a non-resorbable material, such as a siliconepatch or plastic patch, and/or a device such as a vascular catheterand/or other type of medical device which may optionally be inserted tothe body.

The transglutaminase layer can be applied to the first gelatin layersuch that it is noncoextensive with the first gelatin layer and/or willbe noncoextensive with the second gelatin layer upon application of thesecond gelatin layer. For example, the transglutaminase layer can occupyabout 5% to about 95% of the surface area of the first gelatin layerand/or about 5% to about 95% of the surface area of the second gelatinlayer. The transglutaminase can be applied to the gelatin layer in asingle spot or as a series of spots on the gelatin layer such that thetotal surface area of the transglutaminase spots occupies about 5% toabout 95% of the surface area of the first gelatin layer and/or about 5%to about 95% of the surface area of the second gelatin layer.

Such a spot or spots of transglutaminase can have any geometric shape,e.g., filled or unfilled circles, rectangles, triangles, lines,amorphous shapes, or combinations thereof. Such spots can be applied tothe first gelatin layer in an ordered or random pattern. A plurality ofspots can form any of a variety of shapes and patterns, such as anarray, a grid, a series of concentric spots (e.g., concentric circles orsquares), an overlapping series of spots (e.g., overlapping circles),spokes emanating from an axis, or any other configuration, provided thatthe total surface area of the transglutaminase is about 5% to about 95%of the surface area of the first gelatin layer and/or about 5% to about95% of the surface area of the second gelatin layer. In general, a largenumber of small spots is preferred over a small number of large spots.For example, a 20.times.20 array of spots generally is preferable over a10.times.10 array of spots occupying the same total surface area.However, the spots can be of any size provided that the total surfacearea of the transglutaminase is about 5% to about 95% of the surfacearea of the first gelatin layer and/or about 5% to about 95% of thesurface area of the second gelatin layer. For example, depending uponthe overall size of the dressing, the spots can be, without limitation,at least about 0.01, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mm or morein diameter, width, or length. In one embodiment, for example, 4circular spots having a diameter of 2-3 mm each can occupy a squarecentimeter of a dressing. A variety of other configurations are withinthe scope of the invention and can readily be utilized by those skilledin the art.

The dressing can optionally be prepared as any of a variety of sizes andshapes. Typically, the dressings are of a size and shape that canreadily be handled by those skilled in the art, typically less than 12″in length along any side, e.g., 1″×1″, 1″×2″, 4″×4″, etc. The moisturelevel of the dressing typically is less than 8% (e.g., less than 7, 6,5, 4, 3, 2, or 1%).

Any of a variety of resorbable materials known to those skilled in theart can be optionally employed in the present invention. For example,the resorbable material can be a proteinaceous substance, such asfibrin, keratin, collagen and/or gelatin, or a carbohydrate substances,such as alginates, chitin, cellulose, proteoglycans (e.g. poly-N-acetylglucosamine), glycolic acid polymers, lactic acid polymers, or glycolicacid/lactic acid co-polymers. For example, the resorbable material canbe a carbohydrate substance. Illustrative examples of resorbablematerials are sold under the tradenames VICRYL™ and DEXON™.

Generally, the various layers of the hemostatic dressing can be affixedto one another by any means known and available to those skilled in theart. For example, optionally and preferably the gelatin layer(s) and/orthe transglutaminase layer(s) is (are) applied as a series ofquick-frozen aqueous solution layers and subsequently lyophilized orfreeze-dried, e.g., after application of each layer, and upon assemblyof the entire dressing. The layers can be applied by any of a variety oftechniques, including spraying, pipetting (e.g., with a multi-channelpipettor), sprinkling, using a mask, electrostatic deposition, using amicrosyringe array system, or dispensing using a dispensing manifoldthat contains ports for producing a high density array.

In certain embodiments of the present invention, when the dressings areprepared using a mold, a release agent, such as sucrose, is applied tothe mold before the first layer of the dressing is applied. In suchembodiments, the hemostatic dressing further comprises a release layer,which contains said release agent.

Alternatively, a physiologically-acceptable adhesive can be applied tothe resorbable material and/or the backing material (when present) andthe gelatin layer(s) and/or the transglutaminase layer(s) subsequentlyaffixed thereto.

In one embodiment of the dressing, the physiologically-acceptableadhesive has a shear strength and/or structure such that the resorbablematerial and/or backing material can be separated from the gelatin layerafter application of the dressing to wounded tissue. In anotherembodiment, the physiologically-acceptable adhesive has a shear strengthsuch that the resorbable material and/or backing material cannot beseparated from the gelatin layer after application of the dressing towounded tissue.

The concentration of gelatin per area of the wound depends upon a numberof factors, including but not limited to the final construction of thebandage, materials employed and so forth.

According to other embodiments of the present invention, there areprovided methods for preparing a hemostatic dressing by optionally andpreferably providing a first layer of gelatin, applying a layer oftransglutaminase to the first layer of gelatin, and applying a secondlayer of gelatin to the layer of transglutaminase, wherein the layer oftransglutaminase is noncoextensive with the first gelatin layer and/ornoncoextensive with the second gelatin layer.

Similarly, other embodiments of the invention include a method forpreparing a hemostatic dressing by providing a resorbable ornonresorbable backing layer having attached thereto a first layer ofgelatin; applying a layer of transglutaminase to said first layer ofgelatin on a side of the gelatin layer that is opposite of the side towhich the resorbable or nonresorbable backing layer is attached; andapplying a second layer of gelatin to the layer of transglutaminase,wherein the layer of transglutaminase is noncoextensive with the firstgelatin layer and/or noncoextensive with the second gelatin layer.

Hemostatic Device

Another exemplary embodiment of the present invention is directed to ahemostatic device, e.g., for hemostasis in a surgical environment of abriskly bleeding patient, which comprises: (i) a porous resorbable ornon-resorbable matrix; (ii) gelatin in powder, particle, or other solidform, and (iii) transglutaminase in powder, particle or other solidform; wherein the gelatin and transglutaminase are incorporated withinsaid matrix.

Another embodiment of the present invention is directed to a hemostaticdevice, e.g., for hemostasis in a surgical environment of a brisklybleeding patient, which comprises: (i) a porous resorbable ornon-resorbable matrix; (ii) gelatin; (iii) a transglutaminase; whereinthe gelatin and transglutaminase are adhered to said matrix.

Other embodiments of the present invention include application of thehemostatic/sealant mixture through accepted methods of sealantapplication. Such methods may optionally include application of themixture as part of a gel, foam, or spray. Application of thehemostatic/sealant mixture using these methods may optionally beaccomplished for example by storing the mixture components separatelyand mixing them immediately prior to application; and/or for exampleoptionally by storing the components together in inactivated form andactivating them immediately prior to application. Inactivated forms ofthe sealant components may optionally be provided as one or more of afrozen solution, a lyophilized powder that requires reconstitution, aspray dried powder that requires reconstitution, and/or any othersuitable form of inactivated sealant mixture.

Hemostatic Device Preparation

According to some embodiments of the present invention, a freeze dryingand/or lyophilizing technique may optionally be applied to adhere or fixthe sealant composition according to the present invention onto thesurface of any catheters, trocars or implants, or indeed any other suchmedical device. This may optionally facilitate hemostasis at thepenetration wound and its closure, which may optionally be useful forarterial catheters/devices for example. Hemostasis after arterialprocedure is critical for patients who have been treated withanti-coagulation medication and who are more prone to bleedingcomplications. The hemostatic composition of the present invention isindependent of blood clotting and so provides additional assistance toprevent excess bleeding.

Use of Device, Composition or Bandage

During use of the hemostatic dressing, device, or agent, the gelatin andthe transglutaminase can be activated at the time the dressing, device,or particle mixture is applied to the wounded tissue by the endogenousfluids (e.g., blood, air, bile, intestinal fluid) of the patientescaping from the hemorrhaging or leaking wound. Alternatively, insituations where fluid loss from the wounded tissue is insufficient toprovide adequate hydration of the protein layers, the gelatin and or thetransglutaminase can be activated by a application of aphysiologically-acceptable liquid (e.g., water, buffer, saline),optionally containing any necessary co-factors and/or enzymes, prior toor upon application of the hemostatic dressing, device, or agent to thewounded tissue.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and the accompanyingdescription.

Referring now to the drawings, FIG. 1 is a schematic block diagram of anexemplary bandage according to the present invention. As shown, abandage 100 preferably features at least one and preferably a pluralityof layers of gelatin 102, shown as two such layers for the purpose ofdescription only and without any intention of being limiting. At leastone layer of transglutaminase 104 is preferably also provided; in thisexample, layer of transglutaminase 104 is shown as being sandwichedbetween the plurality of layers of gelatin 102 for the purpose ofillustration only and without any intention of being limiting. Asuitable backing 106 is also shown, which preferably provides mechanicalstrength to bandage 100. Backing 106 may optionally be implemented as apolyglycolic acid mesh or patch, such as for example provided as Dexon™or Vicryl™.

FIG. 2 shows a frontal view of an exemplary bandage according to thepresent invention, covered with an optional absorbable backing and anoptional plastic wrapping.

FIG. 3 is a schematic block diagram of an exemplary of a hemostaticdevice according to the present invention, incorporating a porousmatrix. As shown, a hemostatic device 300 preferably features at leastone and preferably a plurality of layers of gelatin 302, shown as twosuch layers for the purpose of description only and without anyintention of being limiting. At least one layer of transglutaminase 304is preferably also provided; in this example, layer of transglutaminase304 is shown as being sandwiched between the plurality of layers ofgelatin 302 for the purpose of illustration only and without anyintention of being limiting. A suitable backing 306 is also shown, whichpreferably provides mechanical strength to bandage 300. Backing 306 mayoptionally be implemented as any type of biodegradable material.

Referring now to the below Examples, various compositions according tothe present invention were constructed and tested for their ability toreduce bleeding and to induce hemostasis. The tested compositions werefound to be very strong and to be able to stop bleeding, even arterialbleeding, in an experimental animal.

Example 1 Preparation of Illustrative Adhesive

This Example relates to the preparation of an illustrative, non-limitingadhesive according to the present invention. For this Example, calciumindependent microbial transglutaminase (Lot No. L-04207, Ajinomoto USA,Chicago, Ill.) was used with a specific activity level of 100 U/gm. Alsothe tested gelatin was Gelatin type A, 300 Bloom from porcine skin(Sigma-Aldrich, St. Louis, Mo.).

The following method was used to prepare the illustrative adhesive: 20%w/w gelatin in PBS (phosphate buffered saline; 20 g gelatin into 80 g ofPBS) was prepared. Next a 20% w/v mTG solution was prepared in PBS (1 gmmTG into 5 mL PBS). Then, 5 g of gelatin solution was mixed with 0.5 mLof mTG solution (in other words 10:1 ratio).

FIG. 4 shows the effect of different percentages of gelatin on theadhesive strength of the adhesive. The adhesive strengths were measuredby adhering a porcine skin sample to a second such sample, placing a47.5 g weight on the joint, and then submerging it immediately in waterfor 120 minutes. After the submersion period, tension was applied at 5mm/min to determine the ultimate adhesion strength (Mcdermott et al.Biomacromolecules 2004, 5, 1270-1279).

As demonstrated in FIG. 5, the optimum reactivity level of microbialtransglutaminase is in the range of 50-55° C. At the physiological levelof 37° C., the reactivity level is only about 60% of the optimum level.As such, raising the temperature of the reaction using an exothermicagent would raise the reactivity level and thus speed up the gelatincross-linking. Thus, optionally and preferably an exothermic agent ispart of the present invention.

Calcium may optionally be used as part of such an agent since calciumchloride releases heat when dissolved but not enough heat to damagetissue. Also, as noted above, it is possible that calcium could helpaccelerate the reaction in other ways, independent of its exothermicdissolution.

Optionally a non-toxic exothermic substance may be included in thebandage with one or more cross-linking factors. Alternatively oradditionally, one or more non-reabsorbable exothermic agents mayoptionally be added behind the bandage backing, as described previously.

Example 2 In Vitro Burst Pressure Test

This Example demonstrates the ability of a composition according to thepresent invention to withstand bursting as a proxy for its ability towithstand high-pressure arterial blood flow. A burst pressure system wasdeveloped, as describe below, to mimic high pressure blood flow, withwarm PBS used in the place of blood to put pressure on a wound in aporcine skin sample. Withstanding 200 mmHg of pressure for 2 minutes wasconsidered the success criteria as physiological blood pressure isnearly always lower than 200 mmHg. These burst test results demonstratedthat compositions according to the present invention are suitable fortreatment of blood flow, including high pressure arterial flow.

Most samples (8/10) withstood a pressurization of 200 mmHg for 2minutes. Those that did not pass were likely related to human or systemerror. The average burst pressure was 320±50 mmHg but this number isconservative since samples that did not burst were assigned a numericalvalue of 354 mmHg, as this was the maximum pressure measurable by theexperimental apparatus. These results demonstrate the capability of theadhesive composition according to some embodiments of the presentinvention to be used for hemostatic purposes, even under rigoroustesting conditions.

Materials

Gelatin (type A from porcine, Bloom value 300) was obtained fromSigma-Aldrich (St. Louis, Mo.). The calcium-independent microbialtransglutaminase (mTG) mixture TG-TI was obtained from Ajinomoto and wasused without further purification. This enzyme is reported by themanufacturer to have a specific activity of 100 U/gm. Porcine skintissue was purchased from a local grocery store.

Sample Preparation

The porcine skin was treated with dilute NaOH for 1 h before cuttinginto a disk shape, with a diameter of about 6-6.5 cm. The fat on theskin was removed with a scalpel. A 2-mm hole was punched at the centerof the skin section to simulate a wound. The skin was washed withcopious amounts of water and PBS buffer, and stored in a Petri dish withabout 1 ml PBS buffer to keep the skin wet until use. For allexperiments described herein, Dulbecco's Phosphate Buffered Saline wasused with a pH of 7.4 for the PBS buffer.

Gelatin solution (25% w/w) in PBS buffer was freshly prepared each dayand stored at 65° C. before use. The mTG (20% w/w) stock solution in PBSbuffer was prepared and aliquotted into 2 ml vials, and stored at −18°C. The enzyme solution was thawed at room temperature before use.

The skin surface was touch-dried with a lab tissue wipe before theadhesive was applied. The adhesive was prepared in a 2 ml vial by mixing1 ml gelatin and 0.5 ml mTG. Two different compositions were prepared.Composition “A” used transglutaminase from Ajinomoto, while composition“B” used transglutaminase from (Yiming Biological Products Co. (Jiangsu,China); the preferred product as described above). 0.6 ml of theresultant mixture (an exemplary tissue adhesive composition according tothe present invention) was applied onto the porcine skin over the hole.After applying the adhesive, the skin tissue was incubated at 37° C. for30 min Burst tests were performed immediately after incubation.

Burst Test

The home-built device was equilibrated in warm buffer (˜44° C.) beforeassembly. After quickly assembling the incubated skin into the device,about 50 ml of 42° C. PBS buffer was poured into the device on top ofthe skin tissue. A nitrogen stream was manually controlled to increasethe pressure. The overall procedure for the burst test was as follows:

Step 1—Increase pressure to 200 mmHg and hold for 2 minutes;

Step 2—Increase pressure to 300 mmHg and hold for 2 minutes;

Step 3—Increase pressure to >354 mmHg (maximum pressure measurable).

As controls, pure gelatin solution was applied onto the skin and allowedto gel (i.e., set) at room temperature for 30 min by forming a physicalgel. Gelatin-Warm refers to the use of 42° C. buffer solution that canmelt the physical gelatin gel.

Results

FIG. 6 shows representative burst pressure measurements of tissueadhesives based on composition A. Data are shown for samples #4 and #5in FIG. 6. A summary of the burst test results for composition A isgiven in Table 2, while the full list of samples is shown in Table 3.

TABLE 2 Summary of burst test results for composition A Total number ofsamples tested 10 No burst 5 Burst after Step #2 1 Burst after Step #1 2Burst during Step #1 2 Average burst pressure* 320 ± 50 mmHg *Anumerical value of 354 mmHg (maximum pressure measurable) was adoptedfor “No failure” samples.

TABLE 3 Burst test results of samples of composition A Burst Sample 200300 pressure Burst # mmHg mmHg (mmHg) type Notes 1 2 min — 325 cohesive2 2 min 2 min >354 3 2 min 2 min >354 4 2 min 2 min >354   5^(a) 30 sec— 232 cohesive Device leak 6 2 min 2 min >354 7 2 min 2 min 348 cohesive8 2 min — 250 cohesive 44° C. PBS 9 2 min 2 min >354 10  10 sec — 245cohesive 44° C. PBS Controls: Gelatin- Warm 13  — — 181 melted 42° C.PBS 14  — — 45 melted 37° C. PBS 15  — — 93 melted 16  — — 105 melted17  — — 84 melted 18  — — 45 melted 19  — — 15 melted 20  — — 140 melted^(a)At ~200 mmHg device started to leak. Tightening the device increasedthe pressure but may have also distorted the skin, resulting in adhesivefailure at 232 mmHg.

FIG. 7 shows representative burst pressure measurements of tissueadhesives of composition B. Data are shown for samples #4 and #5 in FIG.7. Results for the full list of samples are shown in Table 4.

TABLE 4 Burst test results of samples of composition B. Burst Sample 200300 pressure Burst # mmHg mmHg (mmHg) type Notes  1* 2 min max 2 2 min 2min max  3* max, 2 min 4 2 min — 315 cohesive 44° C. PBS 5 2 min 2 minmax *The pressures of these samples were inadvertently set above 200mmHg since the pressure was controlled manually and there is no pressurerelease valve.

Example 3 Hemostasis in a Rat Model

This Example provides an initial in vivo demonstration of a gelatin-mTGcomposition according to the present invention for achieving hemostasisin a live animal. The rat was an adult female Syrian Rat.

Materials

A gelatin solution was used which featured 25% w/w gelatin (porcine,type A, 300 bloom from Sigma-Aldrich (St. Louis, Mo.)) in PBS. Thesolution was mixed by mixing heated (50° C.) PBS into gelatin powder asit was manually stirred using a spatula. Prior to application, gelatinsolution was stored in capped 5 mL syringes submerged in a 50° C. waterbath to maintain its liquid phase.

The transglutaminase (mTG) solution featured 20% w/w microbialtransglutaminase (Activa WM, Ajinomoto™) in PBS. The mTG solution wasmaintained at room temperature.

Prior to application, 1 mL of gelatin solution was added to 0.5 mL ofmTG solution in a 2 mL eppendorf tube. The tube was inverted 2-3 timesto mix the solutions and then solution was applied to the wound siteusing a 1 mL pipette tip. This was the experimental solution.

For the control solution, the protocol was repeated but without theaddition of mTG solution, such that gelatin alone was administered.

For both experimental and control applications, pipette tips were cutapproximately ½ cm from the end in order to expand the opening andenable the passage of the viscous gelatin-mTG solution.

Liver Wound

For both the experimental and control applications, the left lobe of theliver was cut using a scalpel in the rostral-to-caudal direction,creating a 1 cm long, ½ cm deep sagittal cut. After approximately 10seconds of bleeding, cotton gauze was used to remove the accumulatedblood immediately prior to application of either the gelatin (control)or gelatin-mTG (experimental) solutions.

First, the experimental solution was applied to a cut on the left sideof the lobe. A gel formed approximately two minutes after applicationand bleeding was completely stopped in less than about 2.5 minutes afterapplication. After 5 minutes, the tissue was vigorously agitated andtension was applied across the wound site using forceps, yet the gelremained intact and the wound closed. FIG. 8 is a photograph showing theformation of the gel and also induction of hemostasis (FIG. 8A shows theentire area while FIG. 8B shows a portion of the area, magnified forfurther details).

Afterward, the control solution was applied to a cut on the right sideof the lobe. No gel formed and the solution was mostly washed out of thewound site by the blood flow. Even after 6-7 minutes, no clot was formedand the liver continued bleeding (FIG. 9A).

The control solution was removed and the experimental solution was thenapplied to the wound site without removing the accumulated blood. Thoughthe accumulated blood observably hindered adhesion of the experimentalsolution to the liver, a gel still formed that greatly slowed blood flowafter about one minute and completely stopped it after 4.5 minutes (FIG.9B). This demonstrated that the composition of the present invention isable to slow blood flow and induce hemostasis even in the presence ofaccumulated blood.

Femoral Artery Cut

The left femoral artery of the rat was severed using a scalpel. Afterapproximately 10 seconds of heavy bleeding, cotton gauze was used toremove the accumulated blood immediately prior to application of thegelatin-mTG (experimental) solution. As the solution was applied, bloodmixed with the experimental gel as it was undergoing gelation. Underthese rigorous conditions, the gel still completely stopped the bleedingin less than three minutes. After 5 minutes, the gel was manually testedusing forceps. Gel was noticeably less stiff and less adherent when itwas mixed heavily with blood but still formed a strong clot over thesevered artery site. FIGS. 10A-D show photographs of the artery as itwas being cut (10A); the cut artery, bleeding profusely (10B);application of the composition of the present invention to the cutartery (10C); and hemostasis, with formation of a biomimetic clot (10D).

The right femoral artery of the rat was then severed using a scalpel.After approximately 10 seconds of bleeding, cotton gauze was used toremove the accumulated blood immediately prior to application of thegelatin-mTG (experimental) solution. Heavy bleeding was observed but wasalmost immediately halted by the gel and bleeding was completely stoppedin less than one minute. The gel held very strongly and the blood thatwas trapped by gel was readily observable. After 5 minutes, the gel wasmanually tested using forceps. It was adhered very strongly to thetissue in the area of the artery, despite the presence of trapped bloodin the formed gel.

Thus, clearly compositions according to the present invention are ableto slow down the rate of bleeding and to induce hemostasis in an in vivomodel, even in the presence of accumulated blood and/or heavy bleeding(as for example from an artery and/or a vascularized organ including butnot limited to liver, stomach, kidneys, heart, lung and/or skin forexample).

Example 4 Hemostasis in a Porcine Model

This example provides an initial in vivo demonstration of a gelatin-mTGcomposition according to the present invention for achieving hemostasisin a large animal model. The potential for hemostasis utility in a largeanimal model was clearly demonstrated.

Materials

The gelatin solution featured 25% w/w gelatin (porcine, type A, 300bloom from Sigma-Aldrich (St. Louis, Mo.)) in PBS (pH 7.4) and wasprepared as described herein. PBS was stirred continuously at 60° C.using a hot plate magnetic stirrer while gelatin powder was graduallyadded. Manual stirring using a glass stick was performed occasionally toincrease the dissolution rate of the powder and to achieve a homogenoussolution. Throughout the entire experiment, the gelatin solution wasstored in a thermal bath adjusted to ˜50° C. to maintain its liquidphase and prevent the formation of a thermoreversible gel.

The mTG solution featured 20% w/w microbial transglutaminase (Activa WM,Ajinomoto™) dissolved in PBS (pH 7.4). It was prepared as follows. Roomtemperature (RT) PBS solution was stirred using a magnetic stirrer andmTG powder was gradually added. Throughout the entire experiment the mTGsolution was kept in a thermal bath adjusted to ˜30° C., except when inactual use.

An adult, female pig weighing 45 kg was put under general anesthesiaprior to the start of the experiment. Throughout the experiment the pigwas ventilated and its vital signs were monitored.

Prior to application to the wound site as described below, thegelatin-mTG solution according to the present invention was prepared andan applicator was used to place the sealant onto the wound site. Severaldifferent applicators were examined as the bandage's supportivematerial. Unless otherwise stated, before its immediate application ontowound site, 6 mL of novel surgical sealant solution were spread over theapplicator and left to cool for 1 min at RT. This sealant-containing padis considered the “bandage prototype”. For the “control bandage”, asimilar protocol was followed, but with the control solution beingspread over the applicator.

Novel Surgical Sealant Solution—A 2:1 gelatin to mTG mixture wasprepared. Unless otherwise stated, the mixture was prepared by adding 2mL mTG solution to 4 mL gelatin solution in a 15 mL tube and the tubewas inverted 5 times to mix the solutions.

Control Solution—For the control solution the procedure described forNovel Surgical Sealant preparation was repeated, except that PBS alonewas used instead of the mTG solution. Accordingly, gelatin was dilutedin a 2:1 ratio with PBS solution (pH 7.4), submerged in a ˜30° C.thermal bath. Unless otherwise stated, the mixture was prepared byadding 2 mL PBS solution to 4 mL gelatin solution in a 15 mL tube andthe tube was inverted 5 times to mix the solutions.

Applicators were used as follows:

1. A 4 cm×4 cm cotton gauze pad.

2. A 4 cm×4 cm disposable plastic backed absorbent pad. The solution wasspread on the plastic, non-absorbing side of the pad.

3. A silicon mold.

4. A 4 cm×4 cm disposable plastic backed absorbent pad placed inside asilicon mold. The solution was spread on the plastic, non-absorbing sideof the pad.

5. A transparent flexible plastic mold with high margins.

6. Direct application of the sealant on the wound site using a syringeor spilling from a 15 mL tube.

Application of the novel surgical sealant in this study was accomplishedby the surgeon manually placing the sealant over the wound site usingdifferent applicators. If needed, cotton gauze was used to remove theaccumulated blood immediately prior to application. Hemostatic pressurewas applied on the inverse side of the bandage for 3 minutes. After 3minutes, the surgeon relieved pressure and wound site was observed forhemostasis. If full hemostasis did not occur, the wound site was closedby accepted surgical hemostatic techniques. Application of the controlsolution followed the same technique, with accepted hemostatictechniques being immediately applied if hemostasis was not observedafter the control bandage was removed.

Gluteal Muscle Wound

The animal was placed in a prone position and the skin above the glutealmuscles was removed. Overall, 7 experiments were conducted in whichhemostasis and tissue adhesion were examined. Unless otherwise stated,in each trial a 3 cm×3 cm square of muscle was cut 2 cm deep into themuscle, using a #15 scalpel. Excess blood was removed from the woundarea as needed and the novel surgical sealant solution or controlsolution were applied as previously described.

Tables 5 and 6 summarize and describe the experimental procedure andresults of each of the experiments. Table 5 relates to hemostasis whileTable 6 relates to tissue adhesive properties.

Turning first to hemostasis, the control solution was applied to a woundsite using a cotton gauze pad (Table 5, Control #1). The controlsolution was applied on the cotton gauze and left to cool for 1 min 20sec prior to its immediate application. The wound site bled only lightlyand complete hemostasis was observed 2 min after application of thecontrol bandage. Though no biomimetic clot was observed at the woundsite, the hemostatic pressure applied to the wound site was sufficientto encourage hemostasis.

The experiment was repeated in a different wound site, with thedifference being that the applicator used was a disposable plasticbacked absorbent pad and the control solution was left to sit for 30 secprior to its application (Table 5, Control #2). The wound site showedvery little bleeding and 2 min after applying the control bandage,complete hemostasis was observed. As in the previous case, this wasprobably due to the hemostatic pressure applied over the site whileapplying the bandage. No biomimetic clot was observed at the wound site.

Due to the small amount of bleeding observed during the first twocontrol experiment, the experiment was repeated, with the exceptionbeing that a deeper, 4 cm deep cut, was made (Table 5, Control #3).Consequently, heavy bleeding was observed. The control solution wasapplied over an absorbent pad and left to sit for 50 s. The surgeonremoved excess blood from the wound area and applied the controlbandage. After 3 min, bleeding decreased but full hemostasis was notobserved.

The control solution was removed from the wound site created at theformer experiment using a cotton gauze pad. Bleeding was still observed.The novel surgical sealant was applied to the wound area to achievehemostasis (Table 5, Sealant #1). The sealant solution was placed overan absorbent pad, left to sit for 1 min and applied over the wound site.After 3 min, complete hemostasis was observed. The sealant formed abiomimetic clot over the wound site. The gel was agitated using forcepsand strong adherence to the tissue was observed. The gel was removedafter applying some force and appeared as a film. Thus, these resultsdemonstrated the hemostatic properties of the composition according tothe present invention.

TABLE 5 Gluteal Muscle Time to RT Heart Application HemostasisExperiment (° C.) Rate Technique Description (min) Results Control 21 99Cotton gauze The wound site showed 2 Note that # 1 pad little bleedingfollowing following the the incision. Prior to incision, littleapplying, control bleeding from solution was placed over the wound areathe applicator and left to was observed. cool for 1 min 20 sec.Hemostasis was achieved, likely by just applying pressure over the woundsite. Control 21 98 disposable The wound site showed 2 Note that # 2plastic backed little bleeding following following the absorbent pad theincision. Control incision, little solution was placed over bleedingfrom the applicator and left to the wound area cool for 30 sec prior towas observed. applying onto the wound Hemostasis was site. achieved,likely by just applying pressure over the wound site. Control 22 98disposable 4 cm deep cut was made. — The surgeon # 3 plastic backedMassive bleeding was applied absorbent pad observed. Control hemostaticbandage was left to cool pressure due to for 50 sec. Prior to its themassive application over the bleeding. After wound site, excess blood 3min, bleeding was removed. decreased but did not stop. Sealant 22 99disposable Control solution was 3 A strong # 1 plastic backed removedfrom the wound biomimetic clot absorbent pad site performed for wasformed control # 3. Excess blood over the wound was removed. The site.Complete sealant was placed over hemostasis and the bleeding wound site.strong adhesion of the sealant were observed.

After demonstrating the hemostasis capacity of the sealant in a gluteralmuscle model, tissue adhesion was examined (Table 6). Surgical incisionswere made to lift a segment of tissue from the muscle bed, opening awound site.

At the first adhesion experiment (Table 6, Sealant #2), the sealant wasdirectly applied over the wound site and the surgeon applied strongimmediate pressure on the upper part of the tissue for 3 min, displacingall of the sealant from the wound site and resulting in no adhesion.

The experiment was repeated with the exception that following theapplication of the sealant, the surgeon applied only moderate pressure(Table 6, Sealant #3). After 3 min it appeared the tissues adhered. Whenthe upper part of the tissue was agitated, a moderate amount ofresistance was experienced to its complete removal.

The experiment was repeated with special care taken to not displace thesealant from the wound site upon application of pressure (Table 6,Sealant #4). On a different wound site, the sealant was applied on bothparts of the tissue and left for 10 sec. Then, the upper side of thetissue was replaced and moderate pressure was applied. After 3 min,strong tissue adhesion was observed. A significant amount of force wasneeded to then separate the adhered tissues.

TABLE 6 Tissue Adhesion RT Heart Application Tissue Experiment (° C.)Rate Technique Description Adhesion Results Sealant 23 99 Direct Excessblood was N/A No sealant # 2 application removed using a remained in thefrom a tube cotton gauze pad. wound site. The sealant was placed overthe wound site and immediate displacing pressure was applied by thesurgeon. Sealant 23 94 Direct Excess blood was + Adhesion was # 3application removed using a observed with from a tube cotton gauze pad.slight resistance. The sealant was placed at the wound site and thesurgeon applied moderate pressure. Sealant 24 95 Direct The sealant +Strong adhesion # 4 application solution was was observed. from a tubeplaced over the Only after wound site, on applying intensive both partsof the force the tissue tissue and left for ~10 was removed. sec. thenthe upper side of the tissue was replaced and very low pressure wasapplied.

Hemostasis in Liver

The pig was placed supine and its liver was exposed through a midlinelaparotomy. Serial cuts were performed to remove progressively deeperbiopsies of the liver, consequently exposing larger blood vessels.Overall, 5 biopsies were preformed. When needed, cotton gauze was usedto remove the accumulated blood immediately prior to application of thecomposition according to the present invention.

For the first series of biopsies, the control bandage was applied withhemostatic pressure on the inverse side of the bandage for 3 minutes.After 3 minutes, the surgeon relieved pressure and the wound site wasobserved for hemostasis. When full hemostasis did not occur, a deeperbiopsy was performed, followed by application of the novel surgicalsealant. Again, the sealant was applied with hemostatic pressure on theinverse side of the bandage for 3 min and then hemostasis was examined.When full hemostasis was observed, a deeper liver biopsy was removed andthe experiment was repeated with the sealant. This demonstrated thehemostasis capability of the sealant for higher blood pressures. Table 7summarizes the experimental procedure and results of each experiment.

A 4 cm deep biopsy was removed from the left lobe of the liver (Table 7,Control #1). The control solution was applied over an absorbent padplaced in a silicon mold and left to sit for 1 min. The control bandagewas applied over the wound site with hemostatic pressure. After 3 min,pressure was removed and no hemostasis was observed.

After no hemostasis was achieved by applying the control bandage, a 1 cmdeeper biopsy was removed and left to bleed for 30 sec. The experimentwas then repeated with the novel sealant (Table 7, Sealant #1). Thenovel sealant was placed on the pad in a silicon mold and after 1 minapplied to the wound site with hemostatic pressure. After 3 min, thepressure was relieved, the prototype bandage was peeled, and hemostasiswas examined. The sealant created a visible biomimetic film. Hemostasiswas achieved but was not complete since the sealant did not cover theentire wound. It was visible that areas covered with the sealant stoppedbleeding. When the biomimetic film was removed after several minutes,bleeding resumed.

Next, a 1 cm deeper biopsy was removed, resulting in heavy bleeding. Theexperiment was repeated with the exception that a silicon mold was usedas the applicator and excess blood was removed prior to application(Table 7, Sealant #2). The surgeon then applied pressure to the woundsite for 3 min. When the surgeon removed his hand, a biomimetic clot wasvisible over the wound site. The pressure of the blood pushing againstthe biomimetic clot was apparent and after several more minutes, bloodbreached from the rim of the biomimetic sealant. The breach was througha side part of the wound site that was not covered by the sealant. Thisindicated that, at this stage, the hemostatic ability of the sealant isreliant on covering the entire wound site.

To avoid breaching, the former experiment was repeated; with theexception that a larger amount of sealant was applied over the woundsite (Table 7, Sealant #3). A 0.5 cm deeper biopsy was removed from theliver lobe. 9 mL sealant was applied over the wound site with pressure.Unfortunately, during application, nearly all of the sealant dripped offto the sides of the wound site, leaving no discernable sealant on thewound site after pressure was applied by the surgeon.

The experiment was repeated (Table 7, Sealant #4). Another 1 cm biopsywas removed and massive bleeding was observed. This time, 15 mL ofsealant was applied over the wound site using a transparent plastic moldwith high margins to keep the sealant in place. The sealant was placedon the applicator and left to cool for 1 min 20 sec. The sealant wasapplied over the wound site and 4 min later, a thick layer of biomimeticclot was observed and complete hemostasis was achieved. 50 min later thetissue was reexamined and hemostasis was still observed. This indicatedthe strong hemostatic capacity of the sealant when sufficient sealant isapplied to a wound site and maintained in place. The formed biomimeticfilm was difficult to remove as it was strongly adhered to the tissuesurface and removal of the film resulted in a small amount of bleeding.

TABLE 7 Hemostasis in Left Liver Lobe Time to RT Heart ApplicationHemostasis Experiment (° C.) Rate Technique Description (min) ResultsControl 25 87 diposable 4 cm biopsy was — Massive # 1 plastic backedremoved. The bleeding absorbent pad control solution after in a siliconwas placed over application. mold the applicator, left No for 1 min andhemostasis applied with or hemostatic biomimetic pressure over the filmwas wound site for 3 observed. min. Sealant 24 86 diposable Another 1 cm3 The novel # 1 plastic backed biopsy was sealant absorbent pad removedand left partially in a silicon to bleed for 30 stopped the mold sec.massive bleeding by creating a biomimetic film. Complete hemostasis wasnot achieved since the sealant did not cover the entire wound. Sealant24 86 A silicon Another 1 cm 3 The sealant # 2 mold biopsy was did notcover removed and the entire excess blood was wound area, removed. Thethough sealant was hemostasis applied with was hemostatic achievedpressure. where the sealant was present. Sealant 24 84 Silicon moldAnother 0.5 cm — All sealant # 3 biopsy was was removed and displacedexcess blood was during the removed prior to application application ofthe process. sealant. 9 mL of sealant were applied. Sealant 24 80Transparent, Another 1 cm 4 A thick layer # 4 flexible biopsy was ofplastic mold removed. 15 mL biomimetic with high sealant were clot wasmargins applied over the formed. applicator, left 1 Complete min 20 secto cool hemostasis and then placed was over the wound achieved. 50 site.min later the tissue was reexamined and hemostasis was still observed.The formed film was hard to remove and after removal some bleedingcontinued.

Hemostasis in Femoral Artery

Next, the ability of the composition of the present invention to inducehemostasis in wounds or trauma to an artery, specifically the femoralartery, was examined. The animal's right femoral artery was exposed.Then, a circular 2 mm longitudinal cut was preformed using a surgicalblade. Massive bleeding was observed and therefore a hemostat was used.Excess blood was removed using a cotton gauze pad immediately prior toapplication of the sealant. About 9 mL Novel Surgical Sealant wereprepared and applied using a syringe over the wound area. After 4 min,the hemostat was gently removed and hemostasis via the sealant wasexamined. A biomimetic clot was observed and complete hemostasis wasreached. Table 9 summarizes the experimental procedure and results ofthis experiment.

TABLE 9 Hemostasis in Femoral Artery Time to RT Heart ApplicationHemostasis Experiment (° C.) Rate Technique Description (min) ResultsSealant 24 96 A syringe 2 mm punch was 4 After removal # 1 performed anda of the hemostat hemostat was used complete to stop the massivehemostasis was bleeding. ~9 mL of observed. The sealant solution sealantcreated a were applied over biomimetic clot the wound site over thewound using a syringe. site that After 4 min the managed to hemostat wasblock the gently removed. massive bleeding.

Example 5 Protocol Effect of Guanidine Hydrochloride on Gelation andCross Linking

This Example relates to the effect of an exemplary denaturant, guanidinehydrochloride (described herein as “GuCl”), on compositions according tosome embodiments of the present invention. A preferred concentrationratio range is as follows: from about 1:2 to about 2:2 GuHCl:gelatin,weight per weight.

Solution Preparation

1) 10 g of GuCl (Fluka, St. Louis, Mo.) was dissolved in 30 mL ofDulbecco's PBS (Biological Industries, Israel) at room temperature (RT).10 g of type A, 300 bloom porcine gelatin powder (Sigma, St. Louis, Mo.)was weighed separately. Gelatin and GuCl solution were then mixed undermoderate stirring to form a homogenous solution. The resulting solutionhad a gelatin:GuCl ratio (w:w) of 1:1.

The molecular weight (MW) of GuCl is 95.53. In this solution, the finalconcentration of GuCl was thus 3.489 M. Final solution was 20% gelatinw/w, but according to volume, equivalent to 25% w/w gelatin solution inPBS.

2) 2 g of GuCl was dissolved in 30 mL of PBS at RT. 10 g of type A, 300bloom porcine gelatin powder was weighed separately. Gelatin and GuClsolution were then mixed under moderate stirring to form a homogenoussolution. Resulting solution had gelatin:GuCl ration (w:w) of 5:1. Finalconcentration of GuCl was 698 mM. Final solution was 23.8% gelatin w/w,but according to volume, equivalent to 25% w/w gelatin solution in PBS.

3) 6 g of GuCl was dissolved in 30 mL of PBS at RT. 10 g of type A, 300bloom porcine gelatin powder was weighed separately. Gelatin and GuClsolution were then mixed under moderate stirring to form a homogenoussolution. Resulting solution had gelatin:GuCl ration (w:w) of 5:3. Finalconcentration of GuCl was 2.09 M. Final solution was 21.7% gelatin w/w,but according to volume, equivalent to 25% w/w gelatin solution in PBS.

4) 4 g of GuCl was dissolved in 30 mL of PBS at RT. 10 g of type A, 300bloom porcine gelatin powder was weighed separately. Gelatin and GuClsolution were then mixed under moderate stirring to form a homogenoussolution. Resulting solution had gelatin:GuCl ration (w:w) of 5:2. Finalconcentration of GuCl was 1.40 M. Final solution was 22.7% gelatin w/w,but according to volume, equivalent to 25% w/w gelatin solution in PBS.

5) 8 g of GuCl was dissolved in 30 mL of PBS at RT. 10 g of type A, 300bloom porcine gelatin powder was weighed separately. Gelatin and GuClsolution were then mixed under moderate stirring to form a homogenoussolution. Resulting solution had gelatin:GuCl ration (w:w) of 5:1. Finalconcentration of GuCl was 2.79 M. Final solution was 20.8% gelatin w/w,but according to volume, equivalent to 25% w/w gelatin solution in PBS.

Addition of mTG

A 20% w/w solution of 1% microbial transglutaminase powder (mTG)(Ajinomoto Activa TI-WM, Japan) in PBS was prepared.

A) 2 mL of each gelatin-GuCl solution was mixed with 1 mL of mTGsolution in clear, 4 mL plastic tubes. mTG solution was injected intothe gelatin-GuCl solution. Each tube was inverted several times and thenallowed to stand.

B) 2 mL of each gelatin-GuCl solution was mixed with 1 mL of mTGsolution in plastic weighing dishes. Mixtures were manually mixed with apipette tip.

C) Gelatin-GuCl solutions were heated to 43° C. and then 2 mL aliquotsof gelatin-GuCl solutions were mixed with 1 mL of mTG solution inplastic weighing dishes. Mixtures were manually mixed with a pipettetip.

D) Gelatin-GuCl solutions 1 (10 g GuCl) and 5 (8 g GuCl) was heated to43° C. and then 2 mL aliquots of gelatin-GuCl solutions were mixed with2 mL of mTG solution in plastic weighing dishes. Mixtures were manuallymixed with a pipette tip.

Results

1) 1:1 gelatin:GuCl solution formed a homogenous solution at RT within 2minutes. Immediately after formation, solution contained many bubbles.After 2 hrs standing at RT, nearly all of the air bubbles had left thesolution. The solution remained in liquid form and appeared clear with ayellow tint, like standard gelatin solutions. After 24 hours, solutionproperties were unchanged and no more bubbles were visible.

2) 5:1 gelatin:GuCl did not form a homogenous solution at RT. Gelatingranules were swollen but did not dissolve, as would normally occur withgelatin in PBS at RT. Solution was heated to 42° C. until it formed asolution. Upon cooling to RT, it formed a gel at approximately 32° C.

3) 5:3 gelatin:GuCl formed a homogenous solution at RT after 5-6 minutesof vigorous stirring. Immediately after formation, solution containedmany bubbles. After 2 hrs standing at RT, nearly all of the air bubbleshad left the solution. The solution remained in liquid form and appearedclear with a yellow tint, like standard gelatin solutions. Solution wasin liquid form but more viscous than the 1:1 gelatin:GuCl solution.However, it could still be pipetted without difficulty. After 24 hours,solution properties were unchanged and no more bubbles were visible.

4) 5:2 gelatin:GuCl formed a slightly grainy solution at RT after 10minutes of vigorous stirring Immediately after formation, solutioncontained many, many bubbles Immediately after formation, the solutionappeared to be very viscous but still in liquid form. However, after 2hrs standing at RT, many air bubbles were still visible in the solutionand the solution was gelatinous. The solution could not easily bepipetted and was too viscous to mix with other solutions. After 24hours, many air bubbles remained in the solution and the solution hadformed a thermoreversible gel.

5) 5:4 gelatin:GuCl formed a homogenous solution at RT after 2-3 minutesof stirring. Immediately after formation, solution contained manybubbles. After 2 hrs standing at RT, nearly all of the air bubbles hadleft the solution. The solution remained in liquid form and appearedclear with a yellow tint, like standard gelatin solutions. The solutionwas in liquid form, more viscous than the 1:1 gelatin:GuCl solution butless than the 5:3 gelatin:GuCl solution, and could be pipetted withoutdifficulty. After 24 hours, the solution properties were unchanged andno more bubbles were visible.

mTG Results

A) 2 mL Gelatin-GuCl Solution, 1 mL mTG Solution at RT in 4 mL Tube

1) For the 1:1 gelatin:GuCl solution, a small, gelatinous clump wasformed near the top of the tube after 4 minutes. The clump was removedand an additional 1 mL of mTG solution was added. After 35 minutes, asoft-medium gel was formed. The clump was not thermoreversible, asconfirmed by microwave heating. However, it was also not stronglycohesive as a cross-linked gel would be. The soft-medium gel wasconfirmed, using microwave heating, to be thermally irreversible.

2) The 5:1 gelatin:GuCl solution was too viscous to mix with the mTG.

3) For the 5:3 gelatin:GuCl solution, a small, gelatinous clump wasformed near the top of the tube after 4 minutes. It was not removed.After 20 minutes, a medium gel was formed throughout the solution. Theclump was of a distinctly different consistency than the rest of thegel. As above, though it was not thermoreversible, as confirmed bymicrowave heating, it was also not very cohesive and broke apart easilywhen palpated. The medium gel formed became slightly softer upon heatingin the microwave, but was thermally irreversible.

4) Results for the 5:2 gelatin:GuCl solution were very uneven as thesolution became very viscous during the first few minutes after mTGaddition as a result of thermoreveresible gelation (indicated by controlsolution where mTG was not added). After 15 minutes, a moderately firmgel was formed but it was partially thermoreversible and became muchsofter upon heating.

5) For the 5:4 gelatin:GuCl solution, a small, gelatinous clump wasformed near the top of the tube after 4 minutes. It was not removed.After 30 minutes, a medium gel was formed throughout the solution. Theclump was of distinctly different consistency than the rest of the gel.As above, though it was not thermoreversible, as confirmed by microwaveheating, it was also not very cohesive and broke apart easily whenpalpated. The medium gel formed became slightly softer upon heating inthe microwave, but was thermally irreversible.

B) 2 mL Gelatin-GuCl Solution, 1 mL mTG Solution at RT in a Plastic Dish

The gelation time results for the solutions mixed in a plastic dish werenearly identical to those found in the solutions mixed in a 4 mL plastictube: 1:1 gelatin:GuCl formed a soft-medium gel after 35 minutes, 5:3gelatin:GuCl formed a medium gel after 20 minutes, 5:4 gelatin:GuClformed a medium gel after 30 minutes.

However, the gelatinous clumps observed when the mTG was injected intothe gelatin-GuCl solutions was not observed in these experiments.

C) 2 mL gelatin-GuCl solution at 43° C., 1 mL mTG solution in a plasticdish—the results were as follows: for the 1:1 gelatin: GuCl solution, nogel was formed after 35 minutes; for the 5:3 gelatin:GuCl solution, amedium gel was formed after 17 minutes; for the 5:4 gelatin:GuClsolution, a medium gel was formed after 25 minutes.

D) 2 mL gelatin-GuCl solution at 43° C., 2 mL mTG solution in a plasticdish, the results were as follows: for the 1:1 gelatin:GuCl solution, nogel was formed after 25 minutes; for the 5:4 gelatin:GuCl solution, amedium gel was formed after 9 minutes.

From the above results, it was found that GuCl significantly improvesthe solubility of gelatin in PBS. For concentrations of 25% w/w gelatinin PBS, the addition of GuCl at ratios of 5:4 and 1:1 gelatin:GuCl allowgelatin to dissolve in RT PBS almost immediately. This effect issignificantly decreased at a ratio of 5:3 gelatin:GuCl. Forconcentrations of 25% w/w gelatin in PBS, the addition of GuCL at ratiosof 5:3, 5:4, and 1:1 gelatin:GuCl can maintain the gelatin-GuCl solutionin liquid form indefinitely. At a ratio of 5:2 gelatin:GuCl, thesolution undergoes delayed thermoreversible gelation and forms a fullgel after 2 hours. At a ratio of 2:1, gelatin-GuCl solution:mTGsolution, no gel was formed if the gelatin:GuCl solution ratio is 1:1.At lower GuCl concentrations, cross-linked gels were formed. Gelationtime appears to be dependant on GuCl concentration.

Heating the gelatin-GuCl solution to 43° C. prior to mixture with mTGaccelerates the cross-linking process when the gelatin:GuCl ratio is 5:4and 5:3. This was expected as mTG activity increases with increases inreaction temperature up to 55° C. It is likely that if mTG activity isincreased, gelatin:GuCl solutions will be cross-linked more rapidly bymTG.

At a ratio of 1:1, gelatin-GuCl solution:mTG solution, cross-linked gelswere formed even when the gelatin:GuCl solution ratio is 1:1. Theincrease in mTG amount greatly decreased the gelation time of gels thatdid form gels at a gelatin:GuCl solution:mTG solution ratio of 2:1.Gelation time was observed to be dependant on GuCl concentration. Thiswas expected as well as the GuCl likely denatures a certain amount ofmTG. The mTG that is added above that amount is free to cross-linkgelatin.

Without wishing to be limited by a single hypothesis, it is possiblethat if more mTG enzyme is added, gelatin:GuCl solutions would becross-linked much more rapidly by mTG. This can be accomplished, forexample, optionally by removing the carrier from the mTG powder andforming a concentrated solution of the enzyme itself.

Example 6 Protocol Addition of MgCl₂ to Gelatin—Effect on Gelation andCross Linking

This Example relates to the effect of an exemplary reducing agent,magnesium chloride, on compositions according to some embodiments of thepresent invention. A preferred concentration range for dissolvinggelatin into Magnesium Chloride-PBS solution is preferably from about 2to about 4 M, more preferably from about 2.5 to about 3.5M.

Materials and Methods

Materials

Type A 300 bloom porcine gelatin and MgCl2 powder, −325 mesh wereobtained from Sigma-Aldrich corporation (St. Louis, Mo.). Activata TI-WMmicrobial transglutaminase (mTG) was supplied by Ajinomoto (Japan).Dulbecco's PBS (pH 7.4) was obtained from Biological Industries (KibbutzBeit HaEmek, Israel).

mTG Solution Preparation:

Fresh Activa TI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG)mixture was prepared by dissolving in Dulbecco's PBS to form a 20% w/wsolution. The solution was maintained at room temperature (RT) over thecourse of the experiment.

Gelatin-MgCl2 Solution Preparation:

Gelatin was dissolved in different concentrations of MgCl2 solutions asfollows.

Solution A—5 gr of MgCl2 was dissolved in 15 ml of Dulbecco's PBS to afinal concentration of 3.5 M. The dissolution reaction of MgCl2 isexothermic, reaching to 90° C. The solution was left to cool to RT. 25%gelatin aliquot (w/w) was prepared by dissolving 5 gr of gelatin in 15gr of 3.5M MgCl2 solution and stirring at RT.

Solution B— 2.5 gr of MgCl2 was dissolved in 15 ml of Dulbecco's PBS toa final concentration of 1.75 M. The dissolution reaction of MgCl2 isexothermic, reaching to 63° C. The solution was left to cool to RT. 25%gelatin aliquot (w/w) was prepared by dissolving 5 gr of gelatin in 15gr of 63° C. 1.75M MgCl2 solution and stirring.

Effect of MgCl2 on Gelation of Gelatin:

Gelatin dissolved in MgCl2 solutions in different concentrations wereleft to cool to RT. A thermometer was used to follow the temperature ofeach solution. The appearance and viscosity of the gelatin-MgCl2solutions were assessed as the temperature decreased by observation andpalpating the solution with a glass rod.

MgCl2 Effect on Chemical Cross Linking of Gelatin Solutions Using mTG:

Gelatin-MgCl2 “solution A” was tested for chemical cross linking usingmTG. 1 or 2 ml of 20% w/w mTG solution were mixed with 2 ml ofgelatin-MgCl2 solution in a 4 ml technicon tube. The gelatin-MgCl2solution was added either at RT or preheated to 50° C. using a waterbath. The solutions were mixed by gently stirring with the pipette tipand inverting the tube 4 times and time to gelation was measured. Theappearance and viscosity of the gelatin-MgCl2 solutions after mixturewith mTG were assessed by observation and by palpating the solution witha glass rod. When gel was formed it was tested for thermoreversibilityby heating the gel to 50° C. using a water bath.

Results

Effect of MgCl2 on Gelation of Gelatin:

Solution A—3.5 M MgCl2 solution reduced transition point of gelatin.Gelatin-MgCl2 solution was viscous at RT. The solution appeared ratheropaque and consisted small black particles. These particles are probablymagnesium particles that gone oxidation. Solution B— 1.75 Mgelatin-MgCl2 solution gelled at RT. The transition point was 29° C. Thegel was opaque and consisted few black particles that probably formed byoxidation of Mg.

MgCl2 Effect on Chemical Cross Linking of Gelatin Solutions Using mTG:

MgCl2 effect on cross linking was tested with solution A, as follows. 2ml of RT solution A mixed with 1 ml of mTG solution-Irreversible gel wasproduced after 90 min. The gel was very viscous and somewhat soft. 2 mlof 50° C. heated solution A mixed with 1 ml of mTG solution-Irreversiblegel was produced after 70 min 2 ml of 50° C. heated solution A mixedwith 2 ml of mTG solution-Irreversible gel was produced after 25 min thegel was rather weak. The gel's viscosity increased after heating it in a50° C. water bath.

From the above, it appears that addition of magnesium chloride togelatin solutions decreases the transition point of gelatinsignificantly. It appears that the transition point is inverselyproportional to the magnesium chloride concentration. The transitionpoint is reduced to below RT by the addition of 3.5 M of magnesiumchloride. At 1.75 M of magnesium chloride, the transition point of thegelatin solutions is slightly above RT. The addition of magnesiumchloride to gelatin should be optimized to find the minimumconcentration that reduces the gelatin transition point below RT.

It was also shown that cross linking of gelatin using mTG in thepresence of magnesium chloride is possible. Magnesium chloride has adetrimental effect on the cross linking ratio of gelatin. Adding furtheramounts of mTG may optionally be performed to overcome this effect.

mTG activity at 50° C. is far greater than mTG activity at RT. Thisconfirms the theoretical data and indicates the utility of adding anexothermic element into the gelatin-mTG mixture to ensure a reactiontemperature that is higher than RT.

The exothermic dissolution of magnesium chloride may optionally be usedfor both liquefying gelatin and increasing mTG's activity, from theabove data.

Example 7 Protocol Addition of Hydroquinone to Gelatin—Effect onGelation and Cross Linking

This Example relates to the effect of an exemplary reducing agent,hydroquinone, on compositions according to some embodiments of thepresent invention. Hydroquinone is a naturally occurring, water solublereducing agent. Reducing agents can increase gelatin's solubility,allowing it to remain liquid at room temperature (RT). A preferredconcentration range is preferably determined for dissolving gelatin intohydroquinone-PBS solution at concentration of from about 0.2 to about0.5 M, and more preferably from about 0.3 to about 0.4 M.

Materials and Methods

Materials

Type A 300 bloom porcine gelatin and Hydroquinone (ReagentPlus™, >99%)were obtained from Sigma-Aldrich corporation (St. Louis, Mo.). ActivataTI-WM microbial transglutaminase (mTG) was supplied by Ajinomoto(Japan). Dulbecco's PBS (pH 7.4) was obtained from Biological Industries(Kibbutz Beit HaEmek, Israel).

mTG Solution Preparation:

Fresh Activa TI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG)mixture was prepared by dissolving in Dulbecco's PBS to form a 20% w/wsolution. The solution was maintained at room temperature (RT) over thecourse of the experiment.

Gelatin-Hydroquinone Solution Preparation:

Gelatin was dissolved in different concentrations of Hydroquinonesolutions as follows. Solution A—2.75 gr of hydroquinone was dissolvedin Dulbecco's PBS to a final concentration of 0.5 M. Throughout theexperiment the solution was kept at a sealed beaker, wrapped in aluminumfoil, to avoid its exposure to air and light. 25% gelatin aliquot (w/w)in 0.5 M hydroquinone solution was prepared by mixing 5 gr of gelatinwith 15 gr of 0.5 M hydroquinone solution and stirring.

Solution B— 1.32 gr of hydroquinone was dissolved in Dulbecco's PBS to afinal concentration of 0.4 M. Throughout the experiment the solution waskept at a sealed beaker, wrapped in aluminum foil, to avoid its exposureto air and light. 25% gelatin aliquot (w/w) in 0.4 M hydroquinonesolution was prepared by mixing 5 gr of gelatin with 15 gr of 0.4 Mhydroquinone solution. The mixture was stirred and then heated in a 50°C. water bath.

Effect of Hydroquinone on Gelation of Gelatin:

Solutions of gelatin dissolved in different concentrations ofhydroquinone were either kept at RT or heated at a 50° C. water bath andthen cooled to RT. A thermometer was used to follow the temperature ofeach solution. The appearance and viscosity of the gelatin-hydroquinonesolutions were assessed as the temperature decreased by observation andpalpating the solution with a glass rod.

Hydroquinone Effect on Chemical Cross Linking of Gelatin Solutions UsingmTG:

Gelatin-Hydroquinone “solution B” was tested for chemical cross linkingusing mTG. 1 ml of 20% w/w mTG solution was mixed with 2 ml ofgelatin-hydroquinone solution, heated to 50° C., in a 4 ml technicontube. The solutions were mixed by gently stirring with the pipette tipand inverting the tube 4 times and time to gelation was measured. Theappearance and viscosity of the gelatin-hydroquinone solution aftermixture with mTG was assessed by observation and by palpating thesolution with a glass rod. When gel was formed it was tested forthermoreversibility by heating the gel to 50° C. using a water bath.

Results

Effect of Hydroquinone on Gelation of Gelatin

Solution A—0.5 M of hydroquinone solution at RT did not dissolvegelatin. Gelatin powder soaked in the hydroquinone solution, creating agrainy, brown colored, heterogeneous solution. Solution B— 0.4 M ofhydroquinone solution managed to reduce the transition point of gelatin.Hydroquinone solution did not dissolve gelatin at RT. After heating themixture at 50° C. water bath, gelatin dissolved and a homogenoussolution was obtained. The solution was cooled to RT. At 28° C. gelatinremained soluble, but was very viscous. Gel was formed when cooled toRT. The gel was brown colored.

Hydroquinone Effect on Chemical Cross Linking of Gelatin Solutions UsingmTG:

The effect on cross linking was tested with solution B. 2 ml of solutionB heated to 50° C. was mixed with 1 ml of mTG solution. After 4 minirreversible gel was formed. The gel was strong, resembling cross linkedgel that is formed by mixture of 25% (w/w) of gelatin alone with 20%(w/w) of mTG.

From the above it appears that addition of Hydroquinone to gelatinsolutions decreases the transition point of gelatin. At 0.4 M ofhydroquinone, the transition point of the gelatin solutions is slightlyabove RT (28° C.).

In contrast to other substances that have been tested as methods ofreducing transition point of gelatin, hydroquinone did not have asignificant inhibitory effect on the cross linking of gelatin with mTG.

Hydroquinone may optionally be used to reduce the sol-gel transitiontemperature of gelatin without negatively impacting mTG cross-linking ofgelatin, as demonstrated the above data. This is very desirable for manyembodiments of the present invention, as previously described.

Example 8 Protocol Addition of CaCl₂ to Gelatin—Effect on Gelation andCross-Linking

This Example relates to the effect of an exemplary desiccant, calciumchloride, on compositions according to some embodiments of the presentinvention. A preferred concentration range with regard to dissolvinggelatin into Calcium Chloride-PBS solution is preferably in a range offrom about 1 to about 2 M, to decrease the transition point of gelatin.To create an exothermic reaction that could help dissolve gelatin orincrease mTG activity, approximately 0.2-0.7 g of Calcium Chloride permL of solution is optionally and preferably added for each degreeCelsius increase in temperature above the ambient temperature, thoughthe exact amount depends on several factors.

A gelatin-calcium chloride solution in DPBS was prepared as follows: 4 MCaCl2 stock solution was prepared by dissolving 44.396 g of CaCl2 (97%,MW=110.99, Alfa Aesar, Lancaster) in 100 mL of Dulbecco's PBS(Biological Industries, Israel) under stirring. After dissolution, thesolution reached a peak temperature of 80° C. as a result of theexothermic CaCl2 dissolution reaction.

Solution 1 was prepared as follows. 5 g of type A, 300 bloom porcinegelatin powder (Sigma, St. Louis, Mo.) was weighed. 25% w/w gelatinsolutions in PBS-CaCl2 were formed by adding 15 g of differentconcentration PBS-CaCl2 solutions to the 5 g of gelatin. Gelatin-CaCl2was mixed using stir bar as well as occasional manual stirring todisperse clumps. The CaCl2 concentrations tested were:

a. 2M CaCl2 solution in PBS.

b. 2M CaCl2 solution in PBS.

c. 1M CaCl2 solution in PBS.

For both a & b, 2 mL of the gelatin-CaCl2 solution was mixed with 1 mLof 20% w/w microbial transglutaminase (mTG) powder solution, each in a 4mL plastic tube. The mTG product used (Activa TI-WM, Ajinomoto, Japan)had a specific activity of approximately 100 U/g of mTG product powder.

Solution 2 was prepared as follows. A 20% w/w base solution of mTG wasformed by dissolving 10 g of mTG powder into 40 mL of PBS.

a. A 25% w/w gelatin solution was prepared by dissolving 5 g of gelatinin 15 mL PBS by heating gelatin-PBS mixture in microwave for 5 secondsand then 15 seconds. Solution was immediately stirred after eachmicrowave heating period. The temperature after the second heating was72° C. 3.325 g of CaCl2 was then added to form a 2M CaCl2 solution. Thetemperature after the CaCl2 addition was 74° C. Immediately after CaCl2was dissolved and temperature measured, 2 mL of the gelatin-CaCl2solution was mixed with 1 mL of 20% w/w microbial transglutaminase (mTG)powder solution in a 4 mL plastic tube.

b. 5 g of gelatin powder was mixed with 3.33 g of CaCl2 powder. 30 mL ofPBS were then stirred into this mixture to form a solution. Solutiontemperature upon dissolution reached 42° C. After homogenous solutionwas formed, 2 mL of the gelatin-CaCl2 solution was mixed with 1 mL of20% w/w microbial transglutaminase (mTG) powder solution in a 4 mLplastic tube.

Solution 3 was prepared as follows. A 25% gelatin solution in 2MCaCl2-PBS (1a, above) was allowed to sit for 2 hours. Then, 2 mL ofgelatin-CaCl2 solution were mixed with 2 mL of 20% w/w mTG solution in a4 mL plastic tube.

Solution 4 was prepared as follows. After sitting for two hours, a 25%Gelatin solution in 2M CaCl2-PBS (1b, above) was heated to 43° C. Then,2 mL of gelatin-CaCl2 solution were mixed with a. 1 mL of 20% w/w mTGsolution; or b. 2 mL of 20% w/w mTG solution; each in a 4 mL plastictube.

Results

Solutions were formed at all concentrations of CaCl2. Using a 2M CaCl2solution (1a), a homogenous solution was formed and remained in liquidform. The solution was moderately viscous and contained many airbubbles. Prior to mixture with mTG solution, gelatin-CaCl2 solution wasallowed to sit for 30 minutes to allow bubbles to disperse.

The second 2M solution (1b) was identical to the first except that itrequired more manual stirring to disperse a gelatin clump that had beenformed.

Using a 1M CaCl2 (1c) solution, a homogenous solution was formed butstarted to gel after a few minutes. After 2 hours, a thoroughthermoreversible gel had been formed. However, this gel was much softerthan the thermoreversible gel normally formed by the gelatin at roomtemperature. The solution was too viscous to be mixed with mTG solutionafter half an hour.

After addition of the mTG solution to the 2M gelatin-CaCl2 solutions,the solutions became progressively more viscous over a 20 minute periodbut did not form a cohesive gel.

The heating of gelatin-CaCl2 solutions increased the gelling effect ofthe mTG. The microwave heated solution became progressively moreviscous. After 20 minutes, a very soft, irreversible (as confirmed byheating to 50° C.) gel had been formed. The CaCl2 heated solution becameprogressively more viscous. After 20 minutes, a very soft, irreversible(as confirmed by heating to 50° C.) gel had been formed.

For the gelatin-CaCl2 (2M) solution mixed with 2 mL of 20% w/w mTGsolution, a soft gel was formed after 10 minutes. After 20 minutes, amedium strength gel was formed. After 35 minutes, a medium-firm strengthgel was formed.

For the gelatin-CaCl2 (2M) solution heated to 43° C., after being mixedwith 1 mL of 20% w/w mTG solution, a soft gel was formed after 10minutes and a soft-medium strength gel was formed after 20 minutes.However, after being mixed with 2 mL of 20% w/w mTG solution, a mediumstrength gel was formed after 10 minutes and a medium-firm strength gelwas formed after 20 minutes.

Example 9 Protocol Microwave Drying of Gelatin—Effect on Gelation andCross Linking

This Example examines the effect of drying gelatin in a microwave onsolubility. Increased solubility was observed. An optional but preferredmicrowave radiation range of energy preferably features an overallspecific absorption rate (SAR) of from about 1 to about 100 mW/cubiccentimeter, more preferably of from about 30 to about 60 mW/cubiccentimeter. The method was performed as follows.

Gelatin Preparation and Drying

10 gr. portions of type A, 300 bloom porcine gelatin powder (Sigma, St.Louis, Mo.) were weighed into either 50 mL or 250 mL beakers. Thegelatin was then heated in a microwave at 700 W and 2,450 MHz (Kennedymodel KN-949, China) for the following amounts of time:

Sample A: 30 seconds, 50 mL beaker

Sample B: 60 seconds, 250 mL beaker

Sample C: 120 seconds, 250 mL beaker

Sample D: 180 seconds, 250 mL beaker

Control: Gelatin that has not undergone microwave heating

After heating, 30 mL of Dulbecco's PBS (Biological Industries, Israel)at 37° C. was added to the gelatin and the mixture was stirred at 37° C.For Sample A, the PBS was immediately added after the gelatin wasremoved from the microwave. For the rest of the samples, the gelatin wasallowed to cool to room temperature (RT) prior to the addition of thePBS.

For sample C, after gelatin was mixed with the PBS, part of the mixturewas separated, heated to 50° C. and then mixed with a 20% w/w microbialtransglutaminase (mTG) (Ajinomoto Activa TI-WM, Japan) solutionaccording to a 2:1 gelatin solution:mTG solution ratio.

Microwave Heating of Gelatin/PBS Mixture

In Sample F, the gelatin was not heated in powder form. Rather, thegelatin was poured directly into 30 mL of RT PBS in a 50 mL beaker andthen the mixture was heated in the microwave for 15 seconds twiceconsecutively, with a 5 second pause in between the heating periods.After the heating, it was manually stirred. The gelatin solution wasthen mixed with a 20% w/w mTG solution according to a 2:1 gelatinsolution:mTG solution ratio.

Heating of mTG

20% w/w mTG solution was heated in the microwave for 15 seconds twiceconsecutively, with a 5 second pause in between the heating periods. ThemTG was then added to 25% w/w gelatin solution in a 1:2 mTG:gelatinsolution ratio.

Results

Sample A: The gelatin dissolved easily into the PBS, forming a viscoussolution. When cooled to RT, the gelatin solution formed a firmthermo-reversible gel comparable to the thermo-reversible gel formed bystandard gelatin solutions at RT.

Control: The gelatin did not fully dissolve in the PBS. Though it wasfully soaked in PBS, the gelatin remained very grainy.

Sample B: The gelatin did not fully dissolve in the PBS. Though it wasfully soaked in PBS, the gelatin remained very grainy.

Sample C: The gelatin did not fully dissolve in the PBS. Though it wasfully soaked in PBS, the gelatin remained very grainy. The grainygelatin-PBS mixture was then heated in the microwave for 30 seconds. Thetemperature upon removal from the microwave was 76° C. The mixture wasthen manually stirred to form a homogenous solution. The solution wasallowed to cool to RT and formed a thermo-reversible gel comparable tothe thermo-reversible gel formed by standard gelatin solutions at RT.

When the solution was heated to 50° C. and mixed with mTG, a firm andsticky gel was formed after 3 minutes. This gel was heated for 10seconds in the microwave to confirm its irreversibility. Upon exit fromthe microwave, the gel was stickier but stronger. It appeared to beslightly dry.

Sample D: During heating, the gelatin formed a carbonized bubble. Thebubble was formed inside the gelatin powder by burnt gelatin. A strongburning smell accompanied this occurrence.

Heated Gelatin/PBS Mixture: A liquid solution was formed by heating ofthe gelatin/PBS mixture in the microwave. Addition of mTG to thesolution resulted in a firm irreversible gel.

Heated mTG: The mTG that had been heated in the microwave did notcross-link gelatin.

From the above, it appears that heating gelatin powder in a microwavereduces the moisture content of the gelatin, as indicated by significantreductions in the weight of the gelatin (data not shown). Heatinggelatin powder in the microwave, followed by immediate addition of 37°C. PBS reduces the gelatin dissolution time. However, if the gelatinpowder is cooled to RT, then no improvement in gelatin dissolution timeoccurs.

If microwave heated gelatin is microwave heated after it has been mixedwith PBS, then the solution formed can be cross-linked by mTG. It ispossible to dissolve gelatin in RT PBS and then heat it in a microwaveby heating for 15 s and then again by 15 s. Dissolving it in this waydoes not negatively affect mTG cross-linking.

Dry gelatin will burn if heated in the microwave for more than 2minutes. Heating mTG in the microwave drastically reduces its activity.

Example 10 Effect of Urea on Gelation and Cross Linking of Gelatin

This Example relates to the effect of urea as part of an exemplarycomposition according to the present invention. Urea was found to lowerthe transition point of gelatin solutions. The below data confirms thatit sufficiently lowers the transition point of even high concentrationgelatin solution to below room temperature so that the solution is stillin liquid form at room temperature. Transglutaminase was found to beable to cross-link gelatin even in the presence of urea.

Gelatin Solution Preparation:

Type A 300 bloom porcine gelatin (Sigma, St. Louis, Mo.) was used. 25%and 15% w/w gelatin solutions were prepared by dissolving 50 gr and 30gr of gelatin in 150 ml and 170 ml of Dulbecco's PBS (BiologicalIndustries, Israel), respectively, while stirring on hot plate at 50° C.Gelatin was added to PBS gradually and stirred manually using a glassrod. Gelatin solutions were kept in a water bath at 50° C. over thecourse of the experiment.

Transglutaminase Solution Preparation:

Activa TI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG) mixturewas dissolved in Dulbecco's PBS to form a 20% w/w solution. The solutionwas maintained at room temperature (RT) over the course of theexperiment.

Gelatin-Urea Solution Preparation:

40 g aliquots of 25% or 15% w/w gelatin solutions was transferred to 100ml beakers and stirred at 50° C. Urea (98%, Alfa Aesar, Lancaster, UK)was added to these beakers in different ratios, as detailed in the belowtable:

Gelatin Gelatin:Urea Amount of added Urea % ratio (gr) 25% Solution 11:0 0 (Control) Solution 2 1:1 10 15% Solution 3 1:0 0 (Control)Solution 4 1:0.5 2.65 Solution 5 1:1 5.3 Solution 6 1:1.5 8.025

After urea addition, the gelatin-urea solution was stirred at 50° C. for5 min to ensure homogeneity and then transferred to a 37° C. water bath.

Effect of Urea on Thermo-Reversible Gelation of Gelatin:

Each gelatin-urea solution was removed, in turn, from the 37° C. waterbath and left to cool at RT. A thermometer was used to follow thetemperature decrease of each solution. The appearance and viscosity ofthe gelatin-urea solution were assessed as the temperature decreased byobservation and palpating the solution with a glass rod. Results of thisexperience are described at the following table:

Gelatin Gelatin: % Urea (w/w) ratio Results 25% Solution 1 1:0 At 25°C., the gelatin formed a firm thermo-reversible gel. Solution 2 1:1 At25° C., the solution remained in liquid form. Though slightly viscous,it could be easily pipetted. 15% Solution 3 1:0 At 25° C., the gelatinformed a firm thermo-reversible gel. Solution 4 1:0.5 Attemperatures >26° C., the solution remained significantly more liquidthan gelatin alone. However, at RT (23-25° C.), solution formed gel.Solution 5 1:1 At 25° C., the solution remained in liquid form. Afterrefrigeration at 4° C. and return to 25° C., solution returned to liquidform. Solution 6 1:1.5 At 25° C., the solution remained in liquid form.After refrigeration at 4° C. and return to 25° C., solution returned toliquid form.

Cross Linking of Gelatin-Urea Solution Using mTG:

Gelatin-urea solutions were cross linked using mTG. 1 or 2 ml of 20% w/wmTG solution was manually mixed with 2 ml of gelatin-urea solution in aplastic dish. The gelatin-urea solution was added either at RT orpreheated to 50° C. In some tests, it was heated to 50° C. to facilitatecomparison with gelatin alone, which needs to be heated to be mixed withmTG. The solutions were mixed by gently stirring with the pipette tipand left to cross-link for several minutes. As in examination ofthermo-reversible gel formation, the appearance and viscosity of thegelatin-urea solution after mixture with mTG were assessed byobservation and by palpating the solution with a glass rod. The resultsare summarized in the following table:

Amount Gelatin Gelatin: T ° C. of mTG % Urea of added (w/w) ratiosolution (ml) Results 25% Solution 1 1:0 50° C. 1 Normal cross-linking:gelation after 3 minutes. Solution 2 1:1 RT 1 No gel was formed, evenafter 30 min. 2 Soft, sticky gel was formed after 30 minutes 50° C. 1 Nogel was formed, even after 30 min. 2 Soft, sticky gel was formed after10 min. Firm, slightly brittle gel was observed after 30 min. 15%Solution 3 1:0 50° C. 1 Normal cross-linking: gelation after 3 minutes.Solution 4 1:0.5 50° C. 1 Gelation was observed after 6 min. Completegelation was observed after 20 min. Gel was heated in a 50° C. bath yetremained a firm gel. Solution 5 1:1 RT 1 After 12 min gelation began,creating soft gel. After 30 min the gel appears firm. The gel remainedfirm even after heating in a 50° C. water bath.

The above studies show that addition of urea to gelatin solutionsdecreases the transition point of gelatin significantly. For both 15%and 25% w/w gelatin solutions, the transition point is reduced to belowRT by the addition of urea at ratios of 1:1 urea:gelatin and above. Aturea:gelatin ratios of 0.5:1, the transition point of the gelatinsolutions is slightly above RT. It is likely that a urea:gelatin ratiobetween 0.5:1 and 1:1 will suffice to lower the transition point ofgelatin below RT.

It was also shown that cross-linking of gelatin using mTG in thepresence of Urea is possible. However, urea has a detrimental effect onmTG activity. It appears that this effect is relative to ureaconcentration, such that mTG activity in the presence of urea isinversely proportional to the urea concentration.

Transglutaminase activity at 50° C. was far greater than mTG activity atRT, as would be expected. The addition of urea to gelatin can beoptimized to find the minimum concentration that reduces the gelatintransition point below RT. If a sufficient amount of mTG is added, itshould be able to overcome the detrimental effect of urea.

Example 11 Effect of pH on Gelatin Transition Point

This Example demonstrates the effect of changing the pH of gelatinsolutions on the transition point of those gelatin solutions.

Solution Preparation

58.82 gr. of 99% sodium citrate dihydrate (Alfa Aesar, Lancaster, UK)were dissolved in 100 mL of double distilled water to create a 2M stocksolution of sodium citrate. A base solution of 25% w/w gelatin solutionin Dulbecco PBS (Biological Industries, Israel) was prepared using typeA, 300 bloom porcine gelatin (Sigma, St. Louis, Mo.). The gelatinsolution was continuously stirred and maintained at 50° C. 19.21 gr. ofcitric acid anhydrous (Frutarom, Israel) was dissolved in 50 mL ofdouble distilled water to create a 2M stock solution of citric acid.

pH Measurements

The pH of the solution was measured using a pH meter (Eutech pH510,Singapore) with a glass electrode. pH meter was calibrated prior toexperiment using calibration solutions with pH values of 4.01, 7, and10.01. The accuracy of the pH measurement was determined periodicallyover the course of the experiment. The pH of the 2M sodium citratesolution was 8.54. The pH of the 2M citric acid solution was 1.4.

Addition of Sodium Citrate

A 20 mL aliquot of 25% w/w gelatin solution was separated into a 100 mLbeaker, which was maintained at 50° C. with moderate stirring. Theinitial pH of the gelatin solution was measured to be 4.89. Differentamounts of 2M sodium citrate solution were added to 20 mL gelatinsolutions to form the following solutions:

Solution 1: pH of 5.87-2 mL of sodium citrate solution

Solution 2: pH of 6.55-4 mL of sodium citrate solution

Solution 3: pH of 6.7-6 mL of sodium citrate solution

Each solution was then allowed to cool to RT.

Addition of Citric Acid

A 100 mL aliquot of 25% w/w gelatin solution was separated into a 250 mLbeaker, which was maintained at 50° C. with moderate stirring. Theinitial pH of the gelatin solution was measured to be 5.19.

Different amounts of 2M citric solution were added to gelatin solutionsto form the following solutions:

Solution 1: pH of 3.99

Solution 2: pH of 3.54

Solution 3: pH of 2.72

Solution 4: pH of 2.35

Solution 5: pH of 2.17

Solution 6: pH of 2.04

Solution 7: pH of 1.7

Each solution was then allowed to cool to RT.

Sodium Citrate Results

As sodium citrate was added, the addition formed a cloudy, white clumpin the gelatin solution. Vigorous stirring dispersed the clump, firstinto smaller clumps, and then into a homogenous solution. The homogenoussolution that was formed was cloudy and opaque.

At a pH value of 5.87, the gelatin solution aliquot that was allowed tocool formed a thermo-reversible gel in approximately the same amount oftime that gelatin alone forms a thermo-reversible gel.

At a pH value of 6.55, the gelatin solution aliquot that was allowed tocool formed a thermo-reversible gel very rapidly, in under a minute.This was far faster than gelatin alone.

At a pH value of 6.70, the aliquot formed a thermo-reversible gel almostinstantaneously. After the gelatin-sodium citrate solution was left at50° C. for several minutes, the entire solution formed a gel. Thetransition point had increased to a point above 50° C.

At all pH values, the gels were demonstrated to be thermo-reversible asthey all reverted to liquid form after immersion in 60° C. water

Citric Acid Results

A clear difference in transition point was not observed at pH valuesabove 3.54. At the pH value of 3.54, the gelatin solution remainedliquid from 50° C. until approximately 32° C., at which point a verysticky gel was formed.

At a pH value of 2.72, the transition point was approximately 31° C. andthe gel formed was porous: grainy, with many air bubbles.

At a pH value of 2.04, the transition point dropped to 29° C. The gelformed was more porous than the gel formed at a pH value of 2.72.

At a pH value of 1.7, the transition point dropped to 27-28° C. The gelformed was quite porous.

At all pH values, the gels were demonstrated to be thermo-reversible asthey all reverted to liquid form after immersion in 50° C. water.

However, after the gels were left in the 50° C. water for 30 minutes, agel was formed that did not revert to liquid form. This may haveindicated that the citric acid resulted in cross-linking of gelatinafter 30 minutes at 50° C.

The above shows that lowering the pH of a gelatin solution throughaddition of citric acid can significantly decrease the gel transitionpoint. The addition of citric acid, which decreases the gelatin solutionpH to values down to 2, does not result in cross-linking of the gelatin.Further additions of citric acid to lower the pH below 2 may result incross-linking of the gelatin after 30 minutes at 50° C.

Example 12 Effect of Polyhydric Alcohols on Gelation and Cross Linkingof Gelatin

This Example relates to the effect of polyhydric alcohols such assorbitol on gelatin cross linking.

Materials and Methods

Materials

Type A 300 bloom porcine gelatin and 97% D-sorbitol were obtained fromSigma-Aldrich corporation (St. Louis, Mo.). Glycerol 99% was purchasefrom Frutarom (Israel). Activata TI-WM microbial transglutaminase (mTG)was supplied by Ajinomoto (Japan). Dulbecco's PBS (pH 7.4) was obtainedfrom Biological Industries (Kibbutz Beit HaEmek, Israel).

mTG Solution Preparation:

Fresh Activa TI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG)mixture was prepared by dissolving in Dulbecco's PBS to form a 20% w/wsolution. The solution was maintained at room temperature (RT) over thecourse of the experiment.

Gelatin-Polyhydric Alcohol Solutions Preparation:

Solution A—10 gr. of glycerol was dissolved in 30 ml PBS. 10 gr ofgelatin were then soaked in the glycerol solution for 1.5 hours at RT.After 1.5 hours of soaking, 10 ml of PBS were added and the mixture washeated to 50° C. The mixture was manually stirred until a homogenous,liquid solution (1:1 glycerol:gelatin ratio) formed.

Solution B—27.5 ml of 20% w/w gelatin solution was heated to 50° C.while being stirred. 11 gr glycerol was added to the gelatin solution(2:1 glycerol:gelatin ratio). The glycerol-gelatin solution was thenallowed to cool. The 2:1 glycerol:gelatin solution was allowed to soakfor 1.5 h at 50° C. The solution was then removed and allowed to cool atRT.

Solution C—20% gelatin solution containing glycerol in a 2:1glycerol:gelatin ratio, was heated to 50° C. while being stirred. 11 grof sorbitol was added to form a homogenous solution(glycerol:sorbitol:gelatin ratio of 2:2:1).

Solution D—12 gr of sorbitol was added to 20 ml of 20% w/w gelatinsolution at 50° C. to form a homogenous solution with a sorbitol:gelatinration of 3:1. The solution was then allowed to cool.

Solution E—A 5:1 glycerol:gelatin solution was prepared by adding 25 grof RT glycerol to 20 ml of 20% w/w gelatin solution at 50° C. Themixture was mixed by stirring at 50° C.

Solution F—20 gr of sorbitol was added to 20 ml of 20% gelatin solution,resulting in a 5:1 sorbitol:gelatin ratio. The mixture was mixed at 50°C. The mixture was then cooled at RT.

Effect of Polyhydric Alcohol on Gelation of Gelatin:

As prepared with regard to the above description, 2 ml of gelatinsolutions with polyhydric alcohols (see solutions A-F above) wereremoved and left to cool to RT. A thermometer was used to determine thetemperature of each solution. The appearance and viscosity of thegelatin-polyhydric alcohol solutions were assessed as the temperaturedecreased by observation and palpating the solution with a glass rod.

Effect of Polyhydric Alcohols on Cross Linking of Gelatin SolutionsUsing mTG:

Gelatin-polyhydric alcohol solutions were tested for chemical crosslinking using mTG. 1 ml of 20% w/w mTG solution was mixed with 2 ml ofgelatin-polyhydric alcohol solutions in a small plastic dish. Thegelatin-polyhydric alcohol solutions were added either at RT orpreheated to 50° C. using a water bath. The solutions were manuallymixed by gently stirring with the pipette tip and time to gelation wasmeasured. The appearance and viscosity of the gelatin-polyhydric alcoholsolutions after mixture with mTG were assessed by observation and bypalpating the solution with a glass rod. When gel was formed it wastested for thermoreversibility by heating the gel to 50° C. using awater bath.

Results

Effect of Polyhydric Alcohol on Gelation of Gelatin:

Solution A—After soaking in the glycerol, the gelatin particles clumpedtogether to form a very brittle, solid, grainy material. The presence ofglycerol did not seem to slow thermoreversible gelatin gelation at all.A thermoreversible gel was formed in 2-3 minute, as occurs with 20% w/wgelatin solutions without glycerol. At 35° C., the gelatin-glycerolsolution was very viscous, on the verge of gelation. As with the coolingto room temperature, the gelatin-glycerol phase transition was nearlyidentical to that of gelatin alone.

Solution B—As with the 1:1 glycerol:gelatin solution, glycerol at aratio of 2:1 glycerol:gelatin did not lower the transition temperatureof the gelatin. The solution was highly viscous at 35° C. and formed acohesive gel at 33-34° C. Soaking the 2:1 glycerol:gelatin solution for1.5 hours had no effect on the gelatin transition point. Athermoreversible gel started to form at 34° C.

Solution C—At 50° C., the gelatin-glycerol-sorbitol solution was moreviscous than the gelatin-glycerol solution alone and was far moreopaque. When this mixture was cooled, a gel formed at 35° C. Thetransition point was not lowered at all as a result of the sorbitol andglycerol.

Solution D—The gelatin-sorbitol solution started to gel at 40° C.,indicating that high concentrations of sorbitol actually increase thegelatin transition point. At RT, the thermoreversible gel formed was farmore elastic than gelatin gel alone.

Solution E—Even at the very high concentrations of 5:1 glycerol:gelatinratio, the transition temperature of gelatin was not reduced. Athermoreversible gel formed as it would form with gelatin alone.

Solution F—A thermoreversible gel formed at 40° C. Very littledifference was observed between the properties of 3:1 and 5:1sorbitol:gelatin solutions. Both solutions slightly raised thetransition point of gelatin but resulted in extremely sticky and elasticthermoreversible gels.

Effect of Polyhydric Alcohols on Cross Linking of Gelatin SolutionsUsing mTG:

Solution A—The gelatin-glycerol solution was turned into a stiff gel bythe mTG in 3 minutes. The gel formed after 3 minutes was more cohesivethan the gels formed by gelatin and mTG without glycerol over the sametime period. The gel was reheated to 50° C. using a water bath andremained solid, confirming that mTG cross-linking was the mechanism ofgelation.

Solution B—After 3 min, an irreversible gel formed. The gel was reheatedto 50° C. using a water bath and remained solid, confirming that mTGcross-linking was the mechanism of gelation. As noted in the 1:1glycerol:gelatin ratio solution, the presence of glycerol resulted in afirmer gel after 3 minutes. In the 2:1 glycerol:gelatin ratio solution,it was also observed that the formed gel was significantly more brittlethan gels formed with gelatin alone and also noticeably more brittlethan gels formed from the glycerol:gelatin solution with a ratio of 1:1.

Solution C—After 3 minutes, a solid, sticky, very elastic gel wasformed. This gel was not at all brittle and was not easily separated.This was considered a very significant result as the cross-linkedgelatin gel with only glycerol was very brittle. The sorbitol greatlyincreased the elasticity of an otherwise brittle material. The gel wasreheated to 50° C. using a water bath and remained solid, confirmingthat mTG cross-linking was the mechanism of gelation.

Solution E—Results were very similar to results with glycerol at a ratioof 2:1 glycerol:gelatin: the presence of glycerol resulted in theformation of a much more brittle gel than the gels that are formed bygelatin alone. However, after 3 minutes in the presence of mTG, the gelthat was formed was more solid than the gel that is formed by gelatinalone with mTG after 3 minutes.

Solution F—After 3 minutes, a solid yet extremely elastic and sticky gelwas formed by mTG cross-linking of the gelatin-sorbitol solution. Thesorbitol seemed to have no effect on the mTG cross-linking of gelatinoutside of greatly increasing the elasticity and stickiness of themTG-crosslinked gelatin gels.

From the above it was found that the addition of glycerol to gelatindoes not seem to reduce the transition point of gelatin at all. Soakinggelatin in glycerol does not seem to significantly change its propensityfor forming thermoreversible gels. The presence of glycerol seems toresult in stiffer gels after the gelatin solution has been mixed withmTG for 3 minutes. This may indicate an acceleration of the mTGcross-linking of gelatin when gelatin is in the presence of glycerol.

The presence of high concentrations of glycerol during the mTGcross-linking of gelatin appears to make the resulting cross-linked gelsmore brittle than gels formed by the cross-linking of gelatin alone.Glycerol does seem to accelerate the mTG cross-linking of gelatin.

The addition of sorbitol in concert with glycerol does not reduce thetransition point of gelatin. However, sorbitol greatly increases theelasticity and stickiness of gelatin gels. Sorbitol may be able to beused to increase the elasticity of gelatin gels that are rendered morebrittle by the addition of other substances. Sorbitol does not seem toinhibit the mTG cross-linking of gelatin. Though sorbitol seems toslightly increase the transition point of gelatin, it greatly increasesthe elasticity and stickiness of gelatin gels.

Increasing the glycerol:gelatin ration to 5:1 makes the cross-linkedgelatin gels more brittle than those made using a solution of 2:1glycerol:gelatin but does not have any further effect on the gelatintransition point. The slight cross-linking accelerating effect stilloccurs at this higher glycerol:gelatin ratio but is not more pronouncedthan this effect at glycerol:gelatin rations of 1:1 and 2:1.

The solution with a sorbitol:gelatin ratio of 5:1 increases the gelatintransition point but not more than it is increased by the solution witha sorbitol:gelatin ratio of 3:1. However, the cross-linked gelatin gelformed with the 5:1 solution was even more elastic and sticky. Thisfurther suggests that the amount of sorbitol can be altered to vary theelasticity of a cross-linked gelatin gel.

Example 13 Effect of Spray Drying on Gelation and Cross Linking ofGelatin

This Example relates to the effect of spray drying on gelatin crosslinking. A preferred range for particle size formed by using spraydrying is preferably as follows: from about 20 to about 140 μm, morepreferably from about 60 to about 100 μm (diameter).

Various strategies for particle formation may optionally be considered.One potential strategy is to form easily reconstitutable particle ofgelatin and mTG separately either utilizing specialized dryingtechniques to this end or by including additives and then drying thegelatin and mTG with additives into particles. Another potentialstrategy is to form easily reconstitutable particles that incorporategelatin and mTG together. These particles can be formed just byutilizing specialized drying techniques or by including additives thatimprove the reconstitutability of these particles. Furthermore, theseparticles can be created when the gelatin and mTG have not undergone anycross-linking or after they have undergone partial cross-linking.

Materials and Methods

Materials

Type A 300 bloom porcine gelatin was obtained from Sigma-Aldrichcorporation (St. Louis, Mo.). Activata TI-WM microbial transglutaminase(mTG) was supplied by Ajinomoto (Japan). Dulbecco's PBS (pH 7.4) wasobtained from Biological Industries (Kibbutz Beit HaEmek, Israel). Urea,98% was obtained by Alfa Aesar (Lancaster, UK).

mTG Solution Preparation:

20% (w/w) microbial transglutaminase (mTG) solution was prepared bydissolving 4 gr of mTG with 16 gr of Dulbecco's PBS. The solution wasmaintained at room temperature (RT) over the course of the experiment.

4% (w/w) microbial transglutaminase (mTG) solution was prepared bydissolving 2.04 gr of mTG with 50 gr of Dulbecco's PBS. The solution wasmaintained at room temperature (RT) over the course of the experiment.

Gelatin Solution Preparation:

5% (w/w) gelatin solution was prepared by dissolving 10.52 gr of gelatinpowder in 200 gr of Dulbecco's PBS. The mixture was heated to 50° C. andstirred until a homogenous solution was formed. Solution 1 featured 50ml of 5% gelatin solution. Solution 2—1:1 (w/w) gelatin—mannitolsolution was prepared by adding 2.63 gr of mannitol to 50 ml of 5%gelatin solution. The solution was stirred and kept at 50° C. waterbath. Throughout the experiment the solution was kept in ˜50° C. waterdish to prevent thermoreversible gelation.

Solution 3—1:1 (w/w) gelatin-trehalose solution was prepared bydissolving 2.63 gr of trehalose in 50 ml of 5% gelatin solution, whilestirring and heating to 50° C. Throughout the experiment the solutionwas kept at 50° C.

Solution 4—1:1 (w/w) gelatin-urea solution was prepared by dissolving2.63 gr of urea in 50 ml of 5% gelatin solution, while stirring andheating to 50° C. Throughout the experiment the solution was kept at 50°C.

Solution 5—40 gr of 5% gelatin solution was mixed with 20 gr of 4% mTGsolution. Throughout the experiment the solution was kept at 50° C.

Spray Drying of Gelatin Solutions

For the preparation of spray dried gelatin particles, different 5%gelatin solutions in DULBECCO's PBS were prepared (solutions 1-5).Gelatin solutions were spray dried using a BÜCHI micro spray dryer. Theflow type is co-current with mixing of air and liquid at the nozzlehead. The aspirator rate and inlet temperature were kept constant at100% and 100° C., respectively. The liquid feed rate was variedaccording to the process conditions, affecting the outlet temperature asgiven below.

Solution 1—50 ml of 5% (w/w) gelatin solution was kept heated and spraydried at 15% feed rate, with an outlet temperature of 57° C.

Solution 2—50 ml of 1:1 (w/w) gelatin-mannitol solution was kept solublein a 50° C. water bath. The liquid feed rate was 15% with an outlettemperature of 62° C.

Solution 3—50 ml of 1:1 (w/w) gelatin-trehalose solution was keptsoluble in a 50° C. water bath. The liquid feed rate was 15% with anoutlet temperature of 54° C.

Solution 4—50 ml of 1:1 (w/w) gelatin-urea solution was kept soluble ina 50° C. water bath. The liquid feed rate was set to 15% with an outlettemperature of 57° C. Throughout the experiment the liquid feed rate waschanged to 20% with an outlet temperature of 54° C., to enable theformation of powder.

Solution 5—40 ml of 5% gelatin solution mixed with 4% mTG solution wasspray dried in a 20% liquid feed rate and 56° C. outlet temperature.Throughout the experiment the solution was kept in a 37° C. water dish.

Effect of Spray Drying on Gelation of Gelatin Solutions:

Spray dried gelatin powders were dissolved in 4 ml vials usingDulbecco's PBS and mixed by inverting the tube 4 times. The precipitantsolutions were heated at 50° C. water bath and manually mixed, untildissolved. The solutions were then left to cool at RT and the appearanceand viscosity of each solution were assessed as the temperaturedecreased by observation and palpating the solution with a glass rod.

Solution 1—0.33 gr of 5% spray dried gelatin solution was dissolved in 1ml of RT Dullbeco's PBS, to a final of 25% (w/w) gelatin. Than, another1 ml of Dulbecco's PBS was added, reducing gelatin content to 12.5%(w/w).

Solution 2—0.33 gr of spray dried 1:1 gelatin-mannitol solution wasdissolved in 1 ml of RT Dulbecco's PBS.

Solution 3—0.33 gr of spray dried 1:1 gelatin-trehalose solution wasdissolved in 1 ml of RT Dulbecco's PBS.

Solution 4—3 gelatin-urea spray drying failed to produce powder.

Solution 5—0.25 gr of spray dried gelatin-mTG solution was dissolved in0.75 ml of 37° C. Dulbecco's PBS.

Effect of Spray Drying on Chemical Cross Linking of Gelatin SolutionsUsing mTG:

Spray dried gelatin powders were dissolved as described in the previoussection and kept at 50° C. water bath. 20% of RT mTG solution was addedin a 2:1 gelatin to mTG ratio and gently mixed using a pipette tip andby inverting the tube 4 times. Time to gelation was measured and theappearance and viscosity of the solutions after mixture with mTG wereassessed by observation and by palpating the solution with a glass rod.When gel was formed it was tested for thermoreversibility by heating thegel to 50° C. using a water bath.

Results:

Spray Drying of Gelatin Solutions

Spray drying of gelatin solutions provided fine white powder indifferent amounts. Solution 1—˜50 ml of 5% gelatin solution provided0.78 gr. Solution 2—˜40 ml of 5% gelatin solution mixed with mannitol in1:1 ratio (w/w) provided 0.73 gr. Solution 3—˜50 ml of 5% gelatinsolution mixed with trehalose in a 1:1 ratio (w/w) provided 1.135 gr.Solution 4—no powder was produced. The experiment was terminated sincegelatin mixed with urea provided a highly viscous paste that could notbe collected. Solution 5—˜40 ml of 5% gelatin solution mixed with 4% mTGsolution provided 1.27 gr.

Effect of Spray Drying on Gelation of Gelatin Solutions:

Solution 1—gelatin powder partially dissolved in RT PBS to a final 25%(w/w) of gelatin, forming a white non-soluble precipitant. After heatingat 50° C. water bath, the powder dissolved, creating a homogenoussolution. When cooled to RT, the solution gelled, another 1 ml of PBSwas added, to decrease gelatin to 12.5% (w/w). The 12.5% gelatinsolution gelled at 26-27° C.

Solution 2—gelatin-mannitol powder partially dissolved in RT PBS to afinal ˜12.5% (w/w) of gelatin (gelatin is expected to be ½ the amount ofthe produced powder, yet the accurate percentage of gelatin is unknown).A white non-soluble precipitant was formed. After heating in 50° C.water bath, the powder dissolved, creating a homogenous solution. Whencooled to RT, the solution gelled at 28-29° C.

Solution 3—gelatin-trehalose powder partially dissolved in RT PBS to afinal ˜12.5% (w/w) of gelatin (gelatin is expected to be ½ the amount ofthe produced powder, yet the accurate percentage of gelatin is unknown).A white non-soluble precipitant was formed. After heating in 50° C.water bath, the powder dissolved, creating a homogenous solution. Whencooled to RT, the solution gelled at 25-26° C.

Solution 4—the solution contained a cross-linking agent and thereforewas examined for cross-linking rather than gelation.

Effect of Spray Drying on Chemical Cross Linking of Gelatin SolutionsUsing mTG:

Solution 1—500 ul of 20% mTG solution was added to 2 ml of 12.5% gelatinsolution. After 4 min. a strong white gel was formed. The gel wasirreversible.

Solution 2—250 ul of 20% mTG solution was added to 1 ml of 12.5% gelatinsolution with mannitol. After 2.5 min a strong white gel was formed. Thegel was irreversible.

Solution 3—250 ul of 20% mTG solution was added to 1 ml of 12.5% gelatinsolution with trehalose. After 3 min a strong white colored gel wasformed. The gel was irreversible.

Solution 4—the mTG-gelatin solution was dissolved in 1 ml of Dulbecco'sPBS heated to 37° C. A white, weak gel was immediately formed preventingthe complete dissolution in PBS. The formed gel was irreversible.

From the above, it appears that spray drying of gelatin solutions ispossible. For example, 5% gelatin solutions can be spray dried,producing fine white powders. Gelatin can be spray dried with mannitoland trehalose in 1:1 (w/w) gelatin to mannitol or trehalose ratio. Spraydried gelatin-mannitol solutions have a higher transition point comparedto spray dried gelatin alone. Spray dried gelatin-trehalose have a lowertransition point compared to spray dried gelatin alone.

Cross linking of spray dried gelatin solutions is possible. Spray dryingof 1:1 (w/w) gelatin-mannitol solutions improved cross-linking. Spraydrying of 1:1 (w/w) gelatin-trehalose solutions did not affectcross-linking.

Spray drying of gelatin solutions mixed with mTG is possible. Theparticles formed can immediately form a gel upon reconstitution.

Example 14 Applicator for Applying Sealant

This Example relates to an exemplary, illustrative applicator forapplying a hemostatic sealant according to some embodiments of thepresent invention. FIG. 11A shows an example of a double syringeapplicator 1700, featuring two syringes 1702 and 1704, for containingeach component of the two component hemostatic sealant. Syringe 1702 mayoptionally include gelatin or a substitute as described herein, whilesyringe 1704 may optionally include transglutaminase or a substitute asdescribed herein. The difference in volume of the vials will reflect therelated ratio of mixing between the two components. The two componentsmay mix in the nozzle 1705. For improved mixing the nozzle 1705 maycontain a whirlpool creating element 1706 (shown in more detail in FIG.11B). Syringe applicator 1700 may optionally be connected to apressurized air system 1708 at the nozzle 1705, to create a sprayeffect. The pressurized air may optionally enter in the proximal ordistal end of the nozzle 1705 according to the desired application.

Example 15 Catheter and Method of Use Thereof

This Example relates to an exemplary, illustrative catheter and methodof use thereof according to some embodiments of the present invention.FIG. 12A shows an example of a vascular insertion point closure where acatheter 1200 preferably features a coating 1202, comprising the sealantcomponents described herein. Coating 1202 optionally and preferablyfeatures at least one gelatin layer 1204, of which two are shown for thepurpose of illustration only and without any intention of beinglimiting. Gelatin layer 1204 may optionally be substituted by anothertype of protein substrate as described herein. Coating 1202 alsooptionally and preferably features at least one transglutaminase layer1206, which again may optionally be substituted by another cross-linkingmaterial as described herein. Coating 1202 is preferably wrapped aroundan vascular introducer sheath 1208, also referred to as a trocar.

The sheath 1208 may optionally be covered by yet another external sheath1209 that creates a mechanical barrier between the dry sealant componentand the body fluids, as shown in FIG. 12B. Once the external sheath 1209is removed, the sealant components are activated by the body fluids tocreate a peri-vascular entry point closure plug.

While the invention has been described with reference to the foregoingdetailed description thereof and preferred embodiments, the foregoingdescription in intended to illustrate and not limit the invention, whichis defined by the scope of the included claims. Other aspects,advantages, and modifications are within the scope of those claims.

What is claimed is:
 1. A hemostatic or body fluid sealing agent whereinsaid agent comprises a composition of gelatin and a transglutaminasecomposition wherein said transglutaminase composition has a specificgelatin crosslinking activity of from 40U to 200 U/gm of gelatin and inwhich the weight ratio of gelatin to transglutaminase composition is ina range of from 1:1 to 300:1; such that said agent when applied to awound site cross links between gelatin chains and endogenous collagen oftissue extracellular matrix to create a barrier to fluid leakage orbleeding.
 2. The agent according to claim 1, wherein saidtransglutaminase composition comprises a plant, recombinant, animal, ormicrobe derived transglutaminase other than blood derived Factor XIII.3. The agent according to claim 1, wherein said animal origin isselected from the group consisting of fish and mammals.
 4. The agentaccording to claim 1, wherein said microbial derived transglutaminase isisolated from one or more of Streptoverticillium aldaccii, Streptomyceshygroscopicus, or Escherichia coli.
 5. The agent according to claim 1,wherein said composition has a pH in a range of from 5 to
 8. 6. Theagent according to claim 1, further comprising a stabilizer or a filler.7. The agent according to claim 1, wherein gelatin is purified to removesalts and wherein said transglutaminase composition comprises arecombinant or animal derived transglutaminase.
 8. The agent accordingto claim 1, wherein said agent induces hemostasis or stasis of otherleaking bodily fluids in said tissue.
 9. The agent according to claim 1wherein bodily fluid is selected from the group consisting of blood,cerebral spinal fluid, intestinal fluid, air, bile, and urine.
 10. Theagent according to claim 1, wherein said agent induces the formation ofa biomimetic clot at a site of a damaged blood vessel.
 11. The agentaccording to claim 1, wherein said wounded site is selected from thegroup consisting of surgically cut tissue and traumatized tissue.
 12. Ahemostatic or body fluid sealing agent wherein said agent comprises acomposition of gelatin and a transglutaminase composition wherein saidtransglutaminase composition has a specific gelatin crosslinkingactivity of from 40U to 200 U/gm of gelatin for use in the treatment ofwounded tissue, and in which the weight ratio of gelatin totransglutaminase composition is in a range of from 1:1 to 300:1.